CN109415752B - Magnetic electrochemical sensing - Google Patents

Abstract

The target analyte detection device includes a housing having a potentiostat and a microcontroller coupled to the potentiostat. The device also includes a substrate having a plurality of electrodes on a first surface of the substrate. A first set of electrodes of the plurality of electrodes defines a first sample detection region. The substrate is removably attached to the housing such that the first set of electrodes is coupled to the potentiostat when the substrate is attached to the housing. The apparatus also includes a magnet assembly coupleable to the second surface of the substrate. The magnet assembly includes a magnet positioned in the magnet assembly such that, upon coupling the magnet assembly to the substrate, a magnetic field from the magnet extends through the substrate and the first set of electrodes into a region above the first sample detection region.

Description

Magnetic electrochemical sensing

Cross Reference to Related Applications

Priority is claimed for U.S. provisional application No. 62/287,719 filed on 27/1/2016, U.S. provisional application No. 62/288,254 filed on 28/1/2016, and U.S. provisional application No. 62/339,519 filed on 20/5/2016, all of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to systems and techniques for detecting analytes using magnetic electrochemical sensing.

Background

Biological samples are typically examined for the presence and prevalence of specific analytes such as peptides, proteins, lipid metabolites, and other small molecules. In some cases, the presence and prevalence of a particular analyte can provide insight into a particular organism or pathogenic process of a subject, the progression of a particular disease, or some other biological condition. In some cases, the presence and prevalence of a particular analyte can provide insight into the composition of a particular substance or material.

Disclosure of Invention

In one aspect, the present invention provides a target analyte detection device that includes a housing containing a potentiostat and a microcontroller coupled to the potentiostat. The apparatus also includes a substrate having a plurality of electrodes on a first surface of the substrate. A first set of electrodes of the plurality of electrodes defines a first sample detection region. The substrate may be removably connected to the housing such that the first set of electrodes is coupled to the potentiostat when the substrate is connected to the housing. The apparatus also includes a magnet assembly coupleable to the second surface of the substrate. The magnet assembly includes a magnet positioned in the magnet assembly such that, upon coupling the magnet assembly to the substrate, a magnetic field from the magnet extends through the substrate and the first set of electrodes into a region above the first sample detection region.

Embodiments of this aspect may include one or more of the following features. In some embodiments, the housing may contain digital-to-analog converter (DAC) circuitry. The output of the DAC may be electrically coupled to the input of the potentiostat. The housing may also contain analog-to-digital converter (ADC) circuitry. An input comprising circuitry may be coupled to an output of the potentiostat. The housing may also contain a microcontroller electrically coupled to the DAC circuit and the ADC circuit. The microcontroller may be configured to provide a voltage signal to an input of the DAC circuit. The microcontroller may be configured to receive the measurement signal from the output of the ADC circuit.

In some embodiments, the device may comprise a plurality of potentiostats. The plurality of electrodes may comprise at least one set of further electrodes. Each set of electrodes may be coupled to a different respective potentiostat of the plurality of potentiostats, and each set of electrodes may define a different respective sample detection area.

In other embodiments, the apparatus may include a multiplexer electrically coupled to the output of each potentiostat of the plurality of potentiostats. The multiplexer may be configured to electrically couple the selected output to an input of the ADC circuit.

In some embodiments, the device can include an aperture plate disposed on a surface of the substrate, the aperture plate having a plurality of apertures. Each of the plurality of wells can be disposed directly on a different respective sample detection region.

In some embodiments, the magnet assembly may include a plurality of magnets. In coupling the magnet assembly to the substrate, each magnet of the plurality of magnets may be positioned adjacent the substrate and aligned with a respective set of electrodes such that the magnetic field extends from the magnet through the substrate and the respective set of electrodes into a region above a sample detection region defined by the respective set of electrodes.

In further embodiments, the substrate may include a card-edge connector (card-edge connector), and the housing may include a card-edge connector receptacle. In some embodiments, the apparatus may further comprise an electronic communication interface. In other embodiments, the electronic communication interface may include: at least one universal serial bus connector or wireless transceiver. In some embodiments, the first set of electrodes may include three separate electrodes. In other embodiments, the first and second electrodes of the three separate electrodes may be a first metal and the third electrode of the three separate electrodes may be a second metal. In some embodiments, the first set of electrodes may consist of two separate electrodes. In some embodiments, the first set of electrodes may comprise interdigitated electrodes.

In some embodiments, the housing may further comprise a power source and a transceiver coupled to the microcontroller. In some embodiments, the device may further include a display coupled to the microcontroller.

In general, in another aspect, the invention provides a method of detecting the presence of a target analyte. The new method includes providing a plurality of magnetic beads to a first fluid sample. The plurality of magnetic beads includes a first binding moiety that specifically binds to a target analyte. The method also includes allowing a plurality of magnetic beads to bind to a target analyte within the first fluid sample and transferring the magnetic beads from the first fluid sample to the second fluid sample. The second fluid sample includes a second binding moiety that specifically binds to the target analyte. The second binding moiety may be bound to an active enzyme or other reporter group. Transferring the magnetic beads may include immersing the sheath in the first fluid sample, placing a magnet within the sheath immersed in the first fluid sample such that the magnetic beads adhere to the sheath, removing the sheath containing the magnet from the first fluid, and immersing the sheath containing the magnet in the second fluid sample.

The method further includes allowing a second binding moiety within the second fluid sample to bind to the target analyte bound to the first binding moiety of the magnetic beads, combining the second fluid sample comprising the plurality of magnetic beads and the second binding moiety with an electron mediator solution (electron mediator solution) to obtain a third fluid sample, and providing the third fluid sample to the sample detection region of the substrate. The sample detection region may be or be disposed on the first electrode. The method also includes exposing the third fluid sample to a magnetic field to maintain a plurality of magnetic beads in the third fluid sample proximate to the first electrode. The first electrode is electrically coupled to a potentiostat. The method further includes inducing a redox reaction between the electron mediator and the active enzyme within the third fluid sample, and monitoring the output of the potentiostat to determine the presence of the analyte of interest in the third fluid sample. The output of the potentiostat is varied by redox reactions.

Embodiments of this aspect can include one or more of the following features. In some embodiments, inducing the redox reaction can include applying an electrical potential to the second electrode such that the redox reaction occurs, wherein the second electrode is electrically coupled to the potentiostat.

In other embodiments, monitoring the output of the potentiostat may comprise measuring a voltage or current from the first electrode. The voltage or current from the first electrode may vary according to the redox reaction. In some embodiments, monitoring the output of the potentiostat may include selecting the output of the potentiostat from a plurality of different potentiostat outputs, providing the selected output to the microcontroller unit, and presenting the selected output on the display. In some embodiments, the active enzyme may comprise horseradish peroxidase (HRP) and the electron mediator solution comprises 3,3', 5' -Tetramethylbenzidine (TMB).

In some embodiments, the target analyte may comprise extracellular vesicles. In some embodiments, the extracellular vesicles may include exosomes. In some embodiments, the analyte of interest may comprise any of CD24, epCAM, CA125, EGFR, HER2, MUC1, CD44v6, CEA, mesothelin, trop2, GPC1, WNT2, grp94, SSTR2, EGFRv3, IDH1-R132, GPA33, KRAS, CD166, CD133, MET, B7H3, CD63, CD9, and CD81 biomarkers.

In some embodiments, the method may further comprise comparing the output of the potentiostat to a reference level to determine whether the output is above or below the reference level, and diagnosing the presence or absence of cancer in the patient based on the comparison.

In some embodiments, the target analyte may comprise an immune cell marker, such as a CD2, CD3, CD45, CD52, HLA-ABC, CD81, CXCL10 or CXCL9 biomarker.

In some embodiments, the method may further comprise comparing the output of the potentiostat to a reference level to determine whether the output is above or below the reference level, and diagnosing whether the patient rejects the organ transplant based on the comparison.

In some embodiments, the first fluid sample may comprise blood or urine. In various embodiments, the target analyte may include a protein, cell, peptide, protein, lipid, toxin, nucleic acid, microorganism, food antigen, or metabolite.

In some embodiments, the method may further comprise combining the food sample with an extraction buffer to provide a first fluid sample, and incubating the food sample with the extraction buffer to extract the target analyte from the food sample.

In general, in another aspect, the invention provides a kit comprising a sample tube, a sample tube cap, an elongated rod comprising a first magnet, and a target analyte detection device, e.g., a device according to the invention. A target analyte detection device includes a housing, a potentiostat in the housing, and a first substrate including a plurality of electrodes. The plurality of electrodes includes a first set of electrodes defining a first sample detection region. The first substrate is configured to be removably connected to the housing such that the first set of electrodes is coupled to the potentiostat when the first substrate is connected to the housing. The target analyte detection device also includes a second magnet configured to be positioned adjacent to the first substrate such that a magnetic field from the magnet extends through the substrate and the first set of electrodes into a region above the first sample detection region.

Implementations of this aspect may include one or more of the following features. In some embodiments, the sample tube cap may include an opening extending into the elongate sheath. The elongated rod may be sized to fit within the elongated sheath.

In some embodiments, a kit can include an extraction buffer solution having a plurality of magnetic beads, a wash buffer solution, a target analyte buffer solution, and an oxidase buffer solution.

In some embodiments, the target analyte detection device may include a digital-to-analog converter (DAC) circuit. The output of the DAC may be electrically coupled to an input of a potentiostat. The target analyte detection device may also include an analog-to-digital converter (ADC) circuit. The input of the ADC circuit may be coupled to the output of the potentiostat. The target analyte detection device may also include a microcontroller electrically coupled to the DAC circuit and the ADC circuit. The microcontroller may be configured to provide a voltage signal to an input of the DAC circuit. The microcontroller may be configured to receive the measurement signal from the output of the ADC circuit. In some embodiments, the target analyte detection device may include a display.

In some embodiments, the kit can further include a second substrate having an additional plurality of electrodes defining a second sample detection region. The second substrate may be configured to be detachably connected to the housing. In other embodiments, the kit may include a third magnet. The third magnet may be configured to be detachably connected to the second substrate.

One or more of the described embodiments may provide various benefits. For example, embodiments of the magnetic electrochemical sensing system can be used to non-invasively examine biological samples for the presence and prevalence of particular analytes, such as peptides, proteins, lipid metabolites, and other small molecules. In some cases, this information may provide insight into a particular organism or pathogenic process, a particular progression of a particular disease, or some other biological condition. In some cases, this information may provide insight into the composition of particular substances.

In some cases, samples can be analyzed without performing a large number of processing techniques (e.g., filtration or centrifugation) that may require specialized equipment. Thus, a user may analyze a sample more easily and/or in a more cost-effective manner. In some cases, a user may quickly examine each sample, such that many samples may be efficiently examined for the presence of one analyte or multiple different analytes. In some cases, a non-professional user may perform the examination on their own without the aid of an experienced technician, without the need for expensive equipment.

Furthermore, in some cases, cell-specific extracellular vesicles (e.g., exosomes, microvesicles, membrane particles, and apoptotic vesicles or vesicles) can be isolated directly from complex media without the need for extensive filtration or centrifugation. Furthermore, the assay can achieve high detection sensitivity by magnetic enrichment and enzymatic amplification. Furthermore, with an electrical detection scheme, the sensor can be miniaturized and expanded for parallel measurements.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise specified, "target analyte" refers to a single target analyte of a particular type or multiple target analytes of the same type.

Unless otherwise indicated, "specific binding" refers to the formation of a bond between a binding moiety and a particular type of analyte.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description and drawings, and from the claims.

Drawings

FIG. 1 is a schematic diagram of an example of a magnetic electrochemical sensing system for examining an analyte.

FIG. 2 is a diagram of an example of using a magnetic electrochemical sensing system.

Fig. 3 is a diagram of another example of an instrument for examining analytes.

Fig. 4A and 4B are diagrams of another example of an instrument for examining an analyte.

Fig. 4C is a schematic view of the instrument shown in fig. 4A and 4B.

Fig. 5A and 5B are diagrams of another example of an instrument for examining an analyte.

Fig. 5C is a schematic view of the instrument shown in fig. 5A and 5B.

Fig. 6A-6C are diagrams of another example of an instrument for examining an analyte.

Fig. 6D is a diagram of an example of a sample card.

Fig. 6E and 6F are diagrams showing an example of the device.

Fig. 7A-7C are diagrams of examples of Graphical User Interfaces (GUIs) of control applications.

FIG. 8 is a diagram of an example of a sample processing tool.

FIG. 9 is a diagram of an example of using the sample processing tool shown in FIG. 8.

FIG. 10 is a diagram of an example of a computer system.

FIG. 11 is a diagram of an example of a miniaturized magnetic electrochemical sensing system (integrated magneto-electrochemical exosome system, iMEX).

Fig. 12A is a schematic diagram of an example of a low-pass filter.

FIG. 12B shows a comparison of measurements between iMEX sensors and commercial systems (SP 200, bio-Logic).

Fig. 13A-13D are circuit diagrams of an iMEX system.

Fig. 14 is a diagram of a packaged iMEX system.

FIG. 15 is a diagram of an example graphical user interface for customization software to interact with an iMEX system.

FIG. 16 is a schematic of an iMEX assay.

FIG. 17 is another schematic of an iMEX assay.

Fig. 18 is a schematic of electrochemical measurements in an iMEX assay.

Fig. 19A shows the effect of bead size on the measurement current.

Fig. 19B shows the effect of magnetic enrichment on the measurement signal.

Fig. 20 shows a signal comparison of three tetraspanin markers (CD 63, CD9, and CD 81) in cancer extracellular vesicles.

Fig. 21 shows a comparison between iMEX and ELISA. Six surface proteins in two ovarian cancer cell lines (OV 90 and OVCA 420) were analyzed.

Fig. 22 shows a comparison between iMEX and ELISA assays when different numbers of extracellular vesicles were incorporated into human plasma.

Figure 23 shows the analysis of surface proteins and their secreted extracellular vesicles in ovarian cancer cells.

Fig. 24 shows the iMEX assay for clinical sample analysis.

Fig. 25 shows analysis of plasma samples from ovarian cancer patients (n = 11) and healthy controls (n = 5) obtained using the iMEX assay.

Figure 26 shows EpCAM, CD24 and CA125 levels in plasma samples from ovarian cancer patients obtained using the iMEX assay.

Fig. 27 shows longitudinal monitoring of response to drug treatment using iMEX assay.

Fig. 28 is a schematic of T cell-derived Extracellular Vesicle (EV) secretory canaliculus in the kidney.

FIG. 29 shows histology of biopsy samples from renal transplant rejection patients.

Fig. 30 shows a schematic of an iMEX system in a 96-well plate format for detection of Jurkat T cell-derived extracellular vesicles, scanning Electron Microscope (SEM) images of EVs captured by CD3 antibody functionalized magnetic beads, titration curves for Jurkat-derived EV detection by iMEX, and a schematic of iMEX.

Fig. 31A shows the measured current when detecting EVs with CD3 expression with the iMEX assay in the discovery group.

Figure 31B shows ROC curves for determining sensitivity, specificity and accuracy of CD3 markers in the discovery group.

Fig. 32A shows the measured current when detecting EVs with CD3 expression with the iMEX assay in the validation set.

Figure 32B shows ROC curves for determining sensitivity, specificity and accuracy of CD3 markers in the validation set.

Fig. 33A: magnetic electrochemical sensing systems (integrated foreign antigen detection systems, iEAT).

Fig. 33B is a graph of an exemplary iet assay technique.

Fig. 34 is a schematic diagram of an iet system.

FIG. 35 shows the baseline comparison between iEAT performance and commercial equipment (SP-200, bio-Logic).

FIG. 36A shows an example of extraction of Ara h1 with 2-ME buffer.

Fig. 36B and 36C show the performance of three extraction buffers for five test antigens.

Fig. 37 shows an example of a heating device.

Fig. 38A shows amperometric measurements of peanut allergen titrations.

Fig. 38B shows the dynamic current response after applying a reduction potential to the sample.

Fig. 39A and 39B show the activity of lyophilized reagents after storage.

Fig. 40A and 40B show production response curves for various allergens.

Fig. 40C shows the intra-assay variation estimated by measuring three different concentrations of standard.

Fig. 40D shows a comparison between the iEAT results and ELISA measurements.

Fig. 40E shows a comparison of signal responses between various target and non-target samples.

Fig. 41A and 41B show the analysis results obtained using the iet system.

Fig. 41C shows an exemplary graphical user interface for a smartphone application interacting with the iet system.

Fig. 41D shows analysis results obtained using the iet system (left), and an exemplary graphical user interface for a smartphone application interacting with the iet system.

Fig. 42 is a flow chart of an example of a method of detecting the presence of a target analyte.

Detailed description of the preferred embodiments

Systems and techniques for detecting analytes using magnetic electrochemical sensing are described. One or more embodiments described herein can be used to identify analytes such as cells, extracellular Vesicles (EVs) (such as microvesicles, membrane particles, apoptotic vesicles or vesicles) or exosomes (e.g., transmembrane and cytoplasmic proteins, mRNA, DNA and microrna), peptides, proteins, lipids, metabolites and other molecules, which are free-floating (e.g., in serum or solution) or expressed on the surface of a biological structure (e.g., on the surface of an extracellular vesicle or cell).

In an example of an embodiment, a sample (e.g., a biological fluid sample, such as blood or urine) is collected from a subject. The sample is processed using magnetic separation such that a particular analyte of interest (e.g., an analyte indicative of a particular organism or pathogenic process, a particular progression of a particular disease, or some other biological condition) is separated and/or aggregated in the vicinity of a sample probe of a measurement instrument. The measuring instrument is then used to study the presence and/or prevalence of these analytes in the sample via electrochemical detection. The resulting information can be used to provide more effective care to the subject (e.g., by enabling a caregiver to diagnose and/or administer treatment in a more informed manner).

In another example of an embodiment, a sample is collected from a substance. The sample is processed using magnetic separation such that a particular analyte of interest (e.g., an analyte indicative of a particular material or antigen) is separated and/or aggregated in the vicinity of a sample probe of the measurement instrument. The measuring instrument is then used to study the presence and/or prevalence of these analytes in the sample via electrochemical detection. The resulting information can be used to provide insight into the composition of matter. As an example, a sample of a food product may be analyzed for the presence of a particular allergen, so that the consumer may make a more informed choice of his diet.

In some cases, samples can be analyzed without the need to perform extensive processing techniques (e.g., filtration or centrifugation) that may require specialized equipment. Thus, a user may analyze a sample more easily and/or in a more cost-effective manner. In some cases, a user may quickly examine each sample, such that many samples may be efficiently examined for the presence of one analyte or multiple different analytes. In some cases, a non-professional user may perform the examination himself without the aid of an experienced technician, without the need for expensive equipment.

An example of a system 100 for examining analytes is schematically shown in fig. 1. System 100 includes a potentiostat 110 electrically coupled to a probe 120. The system 100 also includes an analog-to-digital converter (ADC) 130, a microcontroller unit (MCU) 140, a digital-to-analog converter (DAC) 150, and a magnet assembly 160.

The probe 120 includes three electrodes: a "reference" electrode 122a, a "counter" electrode 122b, and a "working" electrode 122c. During operation of the system 100, each of these electrodes 122a-c is placed in contact with a fluid sample to be analyzed. Magnet assembly 160 (e.g., placed near probe 120) attracts magnetically labeled particles in the fluid sample to electrodes 122a-c (e.g., by introducing a magnetic field extending through the electrodes). In some cases, one or more of the surfaces or electrodes 122a-c may be collectively referred to as a sample detection region.

Potentiostat 110 is electrically coupled to each of electrodes 122a-c and is configured to maintain a predetermined potential difference between working electrode 122c and reference electrode 122a during operation. Further, potentiostat 110 is configured to measure the current induced from working electrode 122c through counter electrode 122 b.

As shown in fig. 1, potentiostat 110 includes two operational amplifiers 112a and 112b. The first operational amplifier 112a is electrically coupled to the working electrode 122c and is configured to maintain a predetermined potential difference between the working electrode 122c and the reference electrode 122a (e.g., by applying a potential to the working electrode 122c as a deviation from the potential of the reference electrode 122 a). In some cases, the potential may be between about-1.65V and about 1.65V. In some cases, the potential may be about-0.1V. The second operational amplifier 112b is electrically coupled to the reference electrode 122a and the counter electrode 122b and is configured as a transimpedance amplifier to convert the current induced from the working electrode 122c through the counter electrode 122b into a voltage signal.

The electrodes 122a-c may be made of various materials. In some cases, working electrode 122c and counter electrode 122b can be partially or entirely made of a first material (e.g., gold), and reference electrode 122a can be partially or entirely made of a second material (e.g., silver or silver chloride). In some cases, different materials may be used for some or all of the electrodes 122a-c. In some cases, the different materials may increase the signal level by increasing the effective surface area of the electrode.

Potentiostat 110 is electrically coupled to ADC 130 such that a voltage signal (indicative of the current passing from working electrode 122c through counter electrode 122 b) is transmitted to ADC 130. The ADC 130 digitizes the voltage signal (e.g., into a digital signal representing the voltage signal) and transmits the digitized voltage signal to the MCU 140 for processing.

The MCU 140 processes the digitized voltage signals. In some cases, MCU 140 may determine a particular characteristic of the sample based on the digitized voltage signal. For example, based on the digitized voltage signal, MCU 140 can determine whether a particular analyte is present in the sample. As another example, based on the digitized voltage signal, the MCU can determine the absolute concentration and/or the relative concentration of the analyte in the sample.

MCU 140 is also configured to control the operation of potentiostat 110 via DAC 150. For example, the MCU 140 may transmit digital control signals to the DAC 150, and the DAC 150 may convert the digital control signals to corresponding analog signals. These analog signals may be used to control the operation of potentiostat 110. As an example, the MCU 140 via the DAC 150 can increase and/or decrease the potential of the working electrode 122c relative to the reference electrode 122a and control the sampling of current from the working electrode 122c through the counter electrode 122b by applying an analog signal to the inputs of the operational amplifiers 112a and 112b.

Although a three-electrode embodiment is described with reference to fig. 1, it will be appreciated that other configurations may be used to measure induced current within the sample. For example, a structure such as a dual working electrode (e.g., an interdigitated electrode array (IDA)) or a dual electrode (e.g., not including a counter electrode) may be used.

An exemplary use of the system 100 is shown in fig. 2. In this example, the system 100 is used to analyze a fluid sample 202 for the presence of an analyte 204.

As shown at 200 in fig. 2, a fluid sample 202 (containing an analyte 204) is mixed with a solution containing magnetic beads 206. Each magnetic bead 206 includes a magnetic core 208 and one or more binding moieties 210 specific for the analyte 204 coated on a surface of the magnetic core 208.

Upon mixing, the analyte 204 is captured by the magnetic beads 206 due to the interaction between the binding moiety 210 and the analyte 204. The sample 202 can be washed to remove unbound analyte 204 (e.g., by magnetically collecting magnetic beads 206, and transferring the collected magnetic beads 206 to fresh sample buffer).

As shown at 240 in fig. 2, the sample 202 is mixed with a solution containing a second molecule 242. Each second molecule 242 includes a binding moiety 244 specific for the analyte 204. Upon mixing, the second molecules 242 are also captured by the magnetic beads 206 due to the interaction between the binding moieties 244 and the analytes 204. The sample 202 may be washed to remove unbound second molecules 242.

As shown at 260 in fig. 2, the sample 202 is mixed with a solution containing a third molecule 262. Each third molecule 262 includes a binding moiety 264 specific to the second molecule 242 and an active enzyme 266. Exemplary active enzymes 266 include oxidases such as horseradish peroxidase (HRP), alkaline phosphatase, or beta-galactosidase. Upon mixing, the third molecules 262 are also captured by the magnetic beads 206 due to the interaction between the binding moieties 264 and the second molecules 242. The sample 202 may be washed to remove unbound third molecules 262.

As shown at 280 in fig. 2, the sample 202 is mixed with a solution of an electron mediator and applied to the probes 120. Depending on the active enzyme 266, different electron mediator solutions may be used. For example, if the active enzyme 266 comprises HRP, the electron mediator solution may comprise water-soluble substrates such as ABTS (2, 2' -diazabibis [ 3-ethylbenzothiazoline-6-sulfonic acid ] -diammonium salt), OPD (o-phenylenediamine dihydrochloride), and/or TMB (3, 3', 5' -tetramethylbenzidine). As another example, if the active enzyme 266 comprises alkaline phosphatase, the electron mediator solution may comprise a water-soluble substrate such as PNPP (p-nitrophenyl phosphate). As another example, if the active enzyme 266 comprises beta-galactosidase, the electron mediator solution may comprise a water-soluble substrate such AS ONPG (o-nitrophenyl-beta-D-galactopyranoside), nap-Gal (naphthol-AS-B1-beta-D-galactopyranosidase), and/or MUm-Gal (4-methyl-umbelliferyl-beta-D-galactopyranoside).

The magnetic beads 206 are attracted toward the probe 120 due to the magnetic field induced by the magnet assembly 160 under the probe 120. In addition, a redox reaction is induced between the electron mediator and the oxidase enzyme due to the potentials induced across the working electrode 122c and the reference electrode 122a of the probe 120. As a result, a current is induced from the working electrode 122c of the probe 120 through the counter electrode 122 b. Accordingly, the current is converted into a voltage signal by the operational amplifier 112a, the voltage signal is digitized by the ADC 130, and the digitized voltage signal is interpreted by the MCU 140.

In general, the current induced in probe 120 may be related to the presence and/or concentration of analyte 204 in sample 202. For example, if the sample 202 contains a relatively high concentration of analyte 204, a greater amount of analyte will be captured by the magnetic beads 206. Accordingly, a greater number of second molecules 242 and third molecules 262 will also be captured by the magnetic beads 206 and brought into proximity with the probes 120. This results in a greater amount of oxidase available for reaction with the electron mediator solution. Therefore, the redox reaction generates a relatively high current at the counter electrode 122 b.

Conversely, if the sample 202 contains a relatively low concentration of the analyte 204, a lesser amount of the analyte 204 will be captured by the magnetic beads 206. Accordingly, a smaller number of second molecules 242 and third molecules 262 will be captured by the magnetic beads 206 and brought into proximity with the probes 120. This results in a smaller amount of oxidase available for reaction with the electron mediator solution. Therefore, the redox reaction produces a relatively low current at the counter electrode 122 b.

Furthermore, if the sample 202 does not contain any analyte 202, substantially no analyte will be captured by the magnetic beads 204. Accordingly, substantially none of the second molecules 242 and the third molecules 262 will be captured by the magnetic beads. Thus, substantially no oxidase enzyme is available for reaction with the electron mediator solution. Therefore, substantially no current is induced at the counter electrode 122 b.

In some cases, MCU 140 may estimate the absolute concentration and/or the relative concentration of analyte 204 based on the sensed current. For example, in some embodiments, the correlation between the induced current and the analyte concentration can be determined empirically (e.g., by obtaining a sample with a known analyte concentration, measuring the induced current resulting from the analytical process, and deriving a function that describes the relationship between the concentration and the induced current). The concentration of the unknown sample can then be estimated by calibrating the observed current measurements according to the determined correlations. In some cases, the correlation between the induced current and the analyte concentration may be different depending on the analyte, the magnetic beads, the second and third molecules, and/or other parameters. Thus, different correlations may be determined for each set of parameters, and selective application may be appropriate to interpret the current measurement.

Typically, the magnetic bead 206 includes one or more internal magnetic cores and an external coating, such as a capping polymer. The core may be a single metal (e.g., fe, ni, co), a bimetal (e.g., fePt, smCo, fePd, feAu), or may be made of ferrite (e.g., fe ₂ O ₃ 、Fe ₃ O ₄ 、MnFe ₂ O ₄ 、NiFe ₂ O ₄ 、CoFe ₂ O ₄ ) And (4) preparing. The magnetic particles may be of nano-or micro-scale in size and may be diamagnetic, ferromagnetic, paramagnetic or superparamagnetic, where the size corresponds to an average diameter or an average length. For example, the magnetic particles may have a size of about 10 μm, about 1 μm, about 5 μmA size of 00nm, about 300nm, or about 100 nm. In some cases, magnetic particles having a size of about 10 μm may be beneficial in reducing the extent of sedimentation during the assay. Other particle sizes are also possible. The outer coating of the particle may increase its water solubility and stability, and may also provide sites for further surface treatment with binding moieties.

Typically, a binding moiety is a synthetic or natural molecule that specifically binds or is otherwise linked to (e.g., covalently or non-covalently binds to or hybridizes to) a target molecule, or is linked to another binding moiety (or, in some embodiments, to an aggregation-inducing molecule). For example, the binding moiety may be a synthetic oligonucleotide that hybridizes to a specifically complementary nucleic acid target. The binding moiety may also be an antibody directed against an antigen or any protein-protein interaction. Furthermore, the binding moiety may be a polysaccharide that binds to the corresponding target. In some embodiments, a binding moiety may be designed or selected to serve as a substrate for a target molecule (e.g., an enzyme in solution) when bound to another binding moiety. Binding moieties include, for example, oligonucleotides, polypeptides, antibodies, and polysaccharides. For example, streptavidin has four sites (binding moieties) per molecule to be recognized by biotin. For any given analyte, e.g., a particular type of cell having a specific surface marker, there are typically many known binding moieties known to those of skill in the relevant art.

In some cases, the system may be implemented as an analytical instrument. As an example, fig. 3 shows an instrument 300 for examining an analyte using magnetic electrochemical sensing. The instrument 300 includes a housing 310, the housing 310 enclosing the potentiostat 110, the ADC 130, the MCU 140 and the DAC 150 shown in fig. 1. The instrument 300 also includes an aperture 320 exposed along the exterior of the housing 300, the probe 120 is positioned along the bottom of the aperture 320, and the magnet assembly 160 is positioned below the probe 120. This configuration enables a user to apply a fluid sample (e.g., a sample that has been processed according to the method described in fig. 2) to the well 320 such that it rests on top of the probe 120 and the presence and/or prevalence of a particular analyte in the fluid sample is determined using the instrument 300. In some cases, the instrument 300 may output information about the analyzed fluid sample onto a display device 330 (e.g., a display screen or display) and/or output information to a computing device 340 for further analysis (e.g., a computer, smartphone, server system, or other computing device).

In the example shown in fig. 3, the instrument includes a single well with a single probe for analyzing a single sample at a time. However, in some cases, the instrument may include multiple different wells and probes, such that multiple samples may be analyzed simultaneously or sequentially. This may be useful because, for example, it enables a user to process multiple samples in a more efficient manner.

As an example, fig. 4A and 4B illustrate the instrument 400 in an assembled view and an exploded view, respectively. As shown in fig. 4A and 4B, instrument 400 includes a housing 410, a number of apertures 420a-h exposed along the exterior of housing 400, and a magnet assembly 160a-h located below each aperture 410 a-h. In addition, instrument 400 includes a plurality of potentiostats 110a-h, each of which is electrically coupled to a respective probe 120a-h positioned along the bottom of a respective well 420 a-h. In a similar manner as described in fig. 3, a user may apply one or more fluid samples (e.g., samples that have been processed according to the method described in fig. 2) to one or more wells 420a-h and determine the presence and/or prevalence of a particular analyte in the fluid sample using instrument 400. Similarly, the instrument 400 may output information about the analyzed fluid sample onto a display device 430 (e.g., a display screen or display) and/or output information to a computing device 440 for further analysis (e.g., a computer, smartphone, server system, or other computing device).

In some cases, each magnetic assembly 160a-h may include one or more individual magnets corresponding to each aperture 420a-h (e.g., an embedded magnet below each aperture 420 a-h.

In some cases, instrument 400 may include a multiplexer to electrically couple the output of the potentiostats to the MCU, so that the MCU can selectively recover signals from each potentiostat and selectively determine the characteristics of each sample in the well.

As an example, the instrument 400 is schematically illustrated in fig. 4C. Instrument 400 includes a number of potentiostats 110a-h (e.g., a total of 8 as indicated by the "x8" designation in fig. 4C), each of which is electrically coupled to a respective probe 120a-h. The instrument 400 also includes the ADC 130, the MCU 140, the DAC 150, and the magnet assemblies 160a-h. Each of these components may operate similarly as described in fig. 1.

However, in this example, instrument 400 includes a multiplexer 450 that electrically couples the outputs of potentiostats 110a-h to MCU 140 (via ADC 130). Multiplexer 450 selects the voltage signal from one of potentiostats 110a-h and forwards the signal to ADC 130 and MCU 140. Thus, a single ADC 130 and MCU 140 can be configured to selectively recover signals from a plurality of different probes 120a-h and selectively determine the properties of each sample in a well. In some cases, multiplexer 450 may be controlled by a user (e.g., via a switch, dial, button, touch screen, or other control mechanism). In some cases, multiplexer 450 may be controlled by MCU 140 (e.g., by a control signal transmitted from MCU 140 to multiplexer 450 indicating a particular selected hole). This may be useful because, for example, it enables the MCU 140 to automatically select wells for analysis (e.g., to automatically collect measurements from wells in a sequential manner or according to some other pattern).

Although the instrument 400 shown in fig. 4 includes eight wells (and eight corresponding probes and potentiostats), this is merely an illustrative example. In practice, the device may include any number of wells, probes, and potentiostats (e.g., one, two, three, four, five, or more).

Further, in some cases, a multiplexer may be used to electrically couple the outputs of a plurality of different probes to a common potentiostat. This may be useful because, for example, it enables a single potentiostat to measure properties of multiple different samples, which may reduce the complexity and/or cost of producing the instrument.

Another example of an instrument 500 is shown in fig. 5A-C. The instrument 500 includes a housing 510, a plurality of apertures 520 exposed along an exterior of the housing 510, and a magnet assembly 160 positioned below each aperture 520. Further, instrument 500 includes a plurality of potentiostats 110, each of which is electrically coupled to a respective probe 120 positioned along the bottom of a respective well 520.

The wells 520 may have a similar arrangement and similar dimensions as a standardized 96-well plate (e.g., a 96-well plate manufactured according to specifications set by the american national standards institute, ANSI, and/or the laboratory automation and screening association, SLAS). This may be useful because, for example, it enables a user to load and unload samples from the instrument 500 using commonly available equipment (e.g., a standardized multichannel pipette).

As shown in fig. 5B, the aperture 520 and the electrode of potentiostat 110 can be implemented using a series of layers. For example, hole 520 may be disposed in top layer 530, and the electrodes of potentiostat 110 may be disposed on bottom layer 540 (e.g., a substrate such as a printed circuit board). The top layer 530 may be positioned over the bottom layer 540 such that each hole 520 is aligned with a corresponding electrode below. This may be useful because, for example, the instrument 500 may be more easily manufactured (e.g., because multiple sets of electrodes may be manufactured using a single PCB manufacturing process).

In some cases, the electrodes of potentiostat 110 may be formed on a substrate, such as a ceramic substrate, a glass substrate, a rigid plastic substrate, a paper substrate, a flexible polymer substrate (e.g., PDMS), a silicone substrate, and/or a PCB. In some cases, the magnet assembly can include one or more magnets positioned relative to the substrate such that a magnetic field from the magnets extends through the substrate (e.g., to attract magnetic beads to the substrate and electrodes formed thereon). In some cases, the magnet assembly may also be formed on a substrate (e.g., the same substrate as one or more electrodes, or a separate substrate).

The instrument 500 is schematically shown in fig. 5C. In a similar manner to that described in fig. 4C, instrument 500 includes a plurality of potentiostats 110, each potentiostat 110 being electrically coupled to a respective probe 120. The instrument 500 also includes an ADC 130, an MCU 140, a DAC 150, and a magnet assembly 160. Similarly, the instrument 500 includes a multiplexer 450 that electrically couples the output of the potentiostat 110 to the MCU 140 (via the ADC 130). Multiplexer 450 selects the voltage signal from one of potentiostats 110 and forwards the signal to ADC 130 and MCU 140. Thus, a single ADC 130 and MCU 140 can be configured to selectively recover signals from multiple different probes 120 and selectively determine the properties of each sample in a well.

The instrument 500 also includes a Programmable Gain Amplifier (PGA) 550 electrically coupled between the multiplexer 450 and the ADC 130. The PGA 500 may be configured to automatically change the amplification gain of the signal from the multiplexer 450 and maximize or otherwise increase the detection dynamic range of the instrument 500.

The instrument 500 also includes a driver 560 electrically coupled between the DAC 150 and the potentiostat 110. Driver 560 may be configured to deliver current to the electrodes of the potentiostat based on the signal received from DAC 150. This may be beneficial, for example, because some DACs may not be able or suitable to deliver current to a large number of devices.

In some cases, the system may be implemented as a portable device. As an example, fig. 6A shows a portable device 600 for detecting an analyte using magnetic electrochemical sensing. The device 600 includes a housing 610 that encloses the ADC 130, MCU 140, and DAC 150. Device 600 also includes battery module 620, communication interface 630, sample interfaces 640a and 640b, and display device 650.

In general, potentiostat 110, ADC 130, MCU 140, and DAC 150 may function in a manner similar to those described in FIG. 1. For example, the ADC 130 may receive a voltage signal from the potentiostat 110 (which corresponds to the current induced from the working electrode 122c through the counter electrode 122 b), digitize the voltage signal, and transmit the digitized signal to the MCU 140 for processing. Similarly, MCU 140 may process the digitized voltage signals to determine the presence and/or prevalence of a particular analyte, and control the operation of the potentiostat via DAC 150.

In this example, the device 600 does not include a sample probe within the housing 610. Rather, the apparatus 600 includes two sample interfaces 640a and 640b (e.g., communication ports or connectors) through which a user can insert a sample card (and one or more corresponding well and magnet assemblies) containing one or more probes.

For example, as shown in fig. 6B, a user may insert a sample card 650a having a single probe (and a single corresponding well and magnet assembly) into sample interface 640a, and the probe may be electrically coupled to a corresponding potentiostat within housing 610. Sample card 650a may be coupled to sample interface 640a, for example, via a card edge connector (e.g., a pin, socket, or socket) included as part of or located on the substrate of sample card 650 a. This enables the substrate to be removably connected to the rest of the apparatus 600 (e.g., the housing and/or other portions of the apparatus 600) such that the electrodes are coupled to the potentiostat when the substrate is connected to the apparatus 600 (e.g., the housing).

As another example, as shown in fig. 6C, a user may insert a sample card 650b having a plurality of probes (and a plurality of corresponding well and magnet assemblies) into sample interface 640b, and each probe may be electrically coupled to a corresponding potentiostat within housing 610. Similarly, the sample card 650b may be coupled to the sample interface 640b, for example, via a card edge connector (e.g., a pin, socket, or receptacle) included as part of or located on the substrate of the sample card 650 b. Similarly, this enables the substrate to be removably connected to the rest of the device 600 (e.g., the housing and/or other portions of the device 600) such that the electrodes are coupled to the potentiostat when the substrate is connected to the device 600 (e.g., the housing).

This may be useful because, for example, it enables a user to customize the device 600 to his needs. Further, this enables a user to partially disassemble the device 600, which may facilitate maintenance, and may be more conveniently stored or transported. In some cases, one or more potentiostats may also be included on the sample card (e.g., rather than within housing 610).

In some cases, the sample card may be constructed of multiple removable components. For example, as shown in fig. 6D, the sample card 650a may have an electrode portion 660a and a sleeve portion 660b containing the magnet assembly 160. The electrode portion 660a can be inserted into the sleeve portion 660b such that the electrode is positioned above the magnet assembly (e.g., such that one or more magnets of the magnet assembly are positioned below each electrode position). Furthermore, the electrode portion 660a may be separate from the sleeve portion 660b, which allows the electrode portion 660a and/or the sleeve portion 660b to be replaced separately (e.g., separating the substrate with the electrodes from the magnet assembly).

In some cases, sample interface 640a and sample interface 640b may each include a socket having contact pins or other electrical contacts arranged to electrically connect to corresponding electrodes of a card edge connector.

In some cases, the apparatus may be configured to automatically select between sample cards inserted into the sample interface and/or between probes on the sample cards using the styluses of sample interfaces 640a and 650 b. For example, each of the sample interfaces 640a and 650b may include a stylus for detecting the presence of a sample card (e.g., a stylus that receives a signal from a sample card when the sample card is inserted), and a stylus for selecting one or probes on the sample card (e.g., a stylus that transmits a signal identifying a particular probe to an inserted sample card).

Communication interface 630 enables device 600 to communicate with other computing devices (e.g., transmit measurement data to other computing devices and/or receive commands from other computing devices). For example, the communication interface 630 can be a communication port or connector (e.g., an electronic communication interface such as a universal serial bus, USB, connector, plug, jack, or receptacle) that provides a communication channel between the device 600 and another computing device. In some cases, communication interface 630 may be a wireless communication interface (e.g., a wireless transceiver, such as a Wi-Fi or bluetooth transceiver) that enables device 600 to wirelessly communicate with other computing devices.

The display device 650 visually presents information to the user. In some cases, the display device 650 may be a Liquid Crystal Display (LCD), a Light Emitting Diode (LED) display, or an Organic Light Emitting Diode (OLED) display. As an example, FIG. 6E shows display device 650 indicating a first concentration of an analyte in a sample (e.g., a relatively safe amount of a particular analyte), and FIG. 6F shows display device 650 indicating a second concentration of an analyte in a sample (e.g., a relatively dangerous amount of a particular analyte).

In some cases, one or more systems described herein may be controlled via a computing device, such as a computer, smartphone, server system, or other computing device. For example, the device 600 may establish a communication channel with a computing device via the communication interface 630 and use the channel to transmit measurement data to the computing device and receive commands from the computing device.

In some cases, the computing device may execute a control application associated with the magnetic electrochemical sensing system. The control application may provide various functions related to the operation of the system. For example, the control application may present a user interface to the user that presents various options and commands of the operating system and receives input from the user selecting particular options and commands. In addition, the control application may process measurement data received from the system (e.g., convert digitized voltage signals into an indication of a current measurement, determine whether a particular analyte is present based on the current, and/or determine a concentration of the particular analyte based on the current).

To illustrate, fig. 7A shows a smartphone 700 that executes a control application associated with a magnetic electrochemical sensing system. The control application presents a Graphical User Interface (GUI) 710 to the user, which Graphical User Interface (GUI) 710 presents various options for configuring the system (e.g., an option to select a particular sample or "channel" for measurement, and an option to enter a particular analyte to be analyzed). In addition, GUI 710 displays measurement information associated with each selected well (e.g., an indication of the current measured in the selected sample, and an indication of the corresponding analyte concentration based on the measurement). In addition, GUI 710 may display a warning or notification to the user (e.g., if the concentration of a particular analyte is high enough to be potentially unsafe, or if the concentration of a particular analyte exceeds a particular threshold or reference value).

As another example, as shown in fig. 7B, the control application may present GUI 720 to the user, which presents more detailed information about a particular sample. For example, if a user uses the system to analyze a particular substance (e.g., food) for several different analytes (e.g., allergens), GUI 720 may summarize the results of these analyses and visually present the results to the user (e.g., in the form of a graph showing the concentration of each allergen with respect to each other). This enables the user to quickly identify the presence and prevalence of some analytes in the sample.

As another example, as shown in fig. 7C, the control application may present a GUI 730 to the user that presents geographic information about the sample. For example, the GUI 730 may display a geographic map and display points of interest identifying the collection locations of some samples. This enables the user to quickly identify the source of a particular sample, for example, he may search for and/or avoid these locations in the future. For example, the user may enter the locations of the various stores and restaurants from which he obtained food samples, and associate the results of the sample analysis with each appropriate location. If the sample from a particular location contains a high concentration of a particular allergen, the GUI 730 may visually indicate to the user (e.g., using a color-coded icon) so that the user may avoid the location in the future. If a sample from a particular location contains a low concentration of a particular allergen, GUI 730 may visually indicate to the user (e.g., using a different colored icon) so that the user can easily identify the location for future access.

In some cases, the magnetic electrochemical sensing system may be provided as part of an assay kit. In addition to the sensing system, the kit may also include materials that facilitate the preparation of a sample for analysis.

For example, as shown in fig. 8, a kit may include sample processing tools, such as sample tubes 800, sleeves 810, and magnetic strips 820.

Sample tube 800 is configured to receive a fluid sample to be analyzed using a sensing system, such as any of the sensing systems described herein. Sample tube 800 is sealed at first end 802a and defines an opening 804b at second end 802b through which a sample may be deposited.

The cannula 810 defines an elongate channel 812. The passageway 812 is sealed at a first end 814a and defines an opening 816 at a second end 814 b. The channel 812 is sized to receive the magnetic strip 820 inserted through the opening 816 (e.g., the diameter of the channel 812 is slightly larger than the diameter of the magnetic strip 820). Cannula 810 also includes a cap 818, with cap 818 configured to physically connect to sample tube 800 (e.g., via threads on each).

The magnetic strip 820 includes a magnet 822 positioned on a tip thereof and is configured to be inserted into the cannula 810 via the opening 816.

Sample tube 800, cannula 810, and magnetic strip 820 may be used to process a sample for analysis by a sensing system (e.g., by collecting magnetic beads from a sample tube, transferring the collected magnetic beads to another sample tube).

As an example, as shown in fig. 9, a user may insert a sample to be analyzed (e.g., a portion of a food product) into a first sample tube 800a containing an extraction buffer. The extraction buffer is used to extract the target analyte (e.g., protein) for subsequent analysis (e.g., by dissolving the protein and/or peptide in the buffer). A variety of substances may be used as extraction buffers, including 2-mercaptoethanol, tris- (2-carboxyethyl) phosphine enhanced with guanidine (TECP/GUA), TECP with N-lauroylsarcosine, phosphate Buffered Saline (PBS) buffer, tris-HCl buffer, and/or ethanol (e.g., 60% ethanol). As an example, a 2-mercaptoethanol buffer solution can increase protein extractability for some samples (e.g., heated or unheated food products) and can be adapted to reduce protein disulfide bonds and cleave intermolecular disulfide bonds (e.g., those between subunits) to allow for subunit separation of proteins such that each peptide migrates into the buffer solution. As another example, odorless tris- (2-carboxyethyl) phosphine may be used to reduce protein and peptide disulfides. As yet another example, guanidine hydrochloride is a strong chaotropic agent and can be used for denaturation and subsequent refolding of proteins. Such strong denaturants can solubilize insoluble or denatured proteins. As yet another example, N-lauroylsarcosine is an anionic surfactant that can be used for solubilization and separation of proteins and peptides. In some cases, a mixed draw buffer solution (e.g., a draw buffer solution containing multiple different types of component buffer solutions) may be used for extraction. This may be beneficial, for example, because it enables extraction in various use cases, simplifies the extraction process, and/or enhances the user experience.

The user then transfers the material extracted from the first sample tube 800a to a second sample tube 800b containing a sample buffer (e.g., PBS or PBS containing 1% bovine serum albumin BSA). The user also adds a solution containing magnetic beads (e.g., magnetic beads coated with binding moieties specific to a particular target analyte, such as a particular allergen) to sample tube 800b. The user mixes the contents of the second sample tube 800b (e.g., by sealing the second sample tube 800b with the cap and sleeve 810 and shaking the second sample tube 800b.

The user separates the magnetic beads (and the captured analyte) by inserting the magnetic strip 820 into the cannula 810. The magnetic beads are attracted to the perimeter of the cannula 810 by the magnet 822 from the strip 820, thereby retaining it. The user then extracts cannula 810, while still inserting magnetic strip 820, thereby removing the magnetic beads (and captured analyte) from second sample tube 800b.

The user inserts cannula 810 (still inserted with magnetic strip 820) into a third sample tube 800c containing an electron mediator solution (e.g., a solution containing TMB, ABTS, OPD, PNPP, ONPG, nap-Gal, and/or Mum-Gal). The user then removes the magnetic strip 820 from the sleeve 810. This releases the magnetic beads from the cannula 810 and disperses them into the third sample tube 800c. In this manner, a user can selectively remove magnetic beads (and any molecules bound to the magnetic beads) from one sample tube and transfer them to another sample tube without the need for additional filtration or centrifugation equipment.

The user also adds a solution containing a second molecule specific for the analyte (e.g., a binding moiety) and a third molecule specific for the second molecule and having active enzymes (e.g., the second molecule 242 and the third molecule 262 described in fig. 2) in a third sample tube 800c. These second molecules 242 and third molecules 262 are bound to magnetic beads. The user shakes the third sample tube 800c and transfers the contents of the third sample tube 800c to a sensing device for analysis (e.g., by depositing the contents of the third sample tube 800c onto a probe of a potentiostat).

In some cases, the kit may further comprise a heating device, for example as shown in fig. 37. The heating device may be configured to heat the sample tube during the extraction process (e.g., by heating the sample tube and its contents at about 60 ℃ for about 2 minutes) to accelerate the extraction process. In some cases, other heating devices (e.g., microwave ovens, or heat lamps) may be used to heat the sample tubes and their contents during extraction.

As described herein, the magnetic beads are attracted toward the probe due to the magnetic field caused by the magnet assembly below the probe. In addition, a redox reaction is induced between the electron mediator and the oxidase due to the potentials induced across the working and reference electrodes of the probe. As a result, a current is induced from the working electrode through the counter electrode of the probe. Accordingly, the current is converted by the operational amplifier into a voltage signal, the voltage signal is digitized by the ADC, and the digitized voltage signal is interpreted by the MCU.

In some embodiments, the user may wash the sample between some or all of the steps described herein. For example, when transferring samples between sample tubes, a user may use the cannula 810 and magnetic strip 820 to collect magnetic beads (and any bound molecules) from the tubes and transfer the beads to a sample tube containing a wash buffer. The user can remove the magnetic strip 820 to disperse the sample into the wash buffer. The user may then reinsert the magnetic strip 820 into the cannula 810 to recollect the magnetic beads and continue with the sample preparation process. In this manner, a user can wash the magnetic beads (e.g., remove unbound molecules) between some or all of the processing steps described herein.

Some embodiments of the subject matter and the operations described in this specification can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. For example, in some embodiments, systems 100, 300, 400, 500, 600, and 700 may be implemented, at least in part, using digital electronic circuitry, or in computer software, firmware, or hardware, or in combinations of one or more of them. In some embodiments, the potentiostat (e.g., potentiostat 110), the ADC (e.g., ADC 130), the MCU (e.g., MCU 140), and/or the DAC (e.g., DAC 150) and/or the digital-to-analog converter (DAC) 150 may be implemented, at least in part, using digital electronic circuitry, or in computer software, firmware, or hardware, or in a combination of one or more of them.

Some embodiments described in this specification can be implemented as one or more groups or modules of digital electronic circuitry, computer software, firmware, or hardware, or combinations of one or more of them. Although different modules may be used, each module need not be different, and multiple modules may be implemented on the same digital electronic circuitry, computer software, firmware, or hardware, or combinations thereof.

Some embodiments described in this specification can be implemented as one or more computer programs, i.e., one or more modules of computer program instructions, encoded on computer storage media for execution by, or to control the operation of, data processing apparatus. For example, the control application described herein with reference to FIG. 7 may be implemented as a computer program. The computer storage medium may be or may be included in a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or a combination of one or more of them. Further, while a computer storage medium is not a propagated signal, a computer storage medium can be a source or destination of computer program instructions encoded in an artificially generated propagated signal. The computer storage medium may also be or be included in one or more separate physical components or media (e.g., multiple CDs, disks, or other storage devices).

The term "data processing apparatus" encompasses all types of instruments, apparatus, and machines for processing data, including by way of example a programmable processor, a computer, a single-chip system, or a combination of multiple of the foregoing or any of the foregoing. The apparatus may comprise special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit). The apparatus can include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, a cross-platform execution environment, a virtual machine, or a combination of one or more of them. The apparatus and execution environment may implement a variety of different computing model infrastructures, such as web services, distributed computing and grid computing infrastructures.

A computer program (also known as a program, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages. The computer program may correspond to a file in a file system, but is not required. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.

Some of the processes and logic flows described in this specification can be performed by one or more programmable processors executing one or more computer programs to perform actions by operating on input data and generating output. The processes and logic flows can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit).

Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and the processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. A computer includes a processor for performing actions in accordance with instructions and one or more memory devices for storing instructions and data. A computer may also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. However, the computer does not necessarily have such a device. Suitable means for storing computer program instructions and data include all forms of non-volatile memory, media and storage devices, including, for example, semiconductor memory devices (e.g., EPROM, EEPROM, flash memory devices, etc.), magnetic disks (e.g., internal hard disk, removable disk, etc.), magneto-optical disks, CD ROMs, and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

To provide for interaction with a user, operations may be implemented on a computer having a display device (e.g., a display or other type of display device) and a keyboard for displaying information to the user and a pointing device (e.g., a mouse, trackball, tablet, touch-sensitive screen, or other type of pointing device) by which the user may provide input to the computer. Other types of devices may also be used to provide for interaction with a user; for example, feedback provided to the user can be any form of sensory feedback, such as visual feedback, auditory feedback, or tactile feedback; and input from the user may be received in any form, including acoustic, speech, or tactile input. In addition, the computer may interact with the user by sending and receiving documents to and from the device used by the user; for example, by sending a web page to a web browser on the user's client device in response to a request received from the web browser.

A computer system may comprise a single computing device, or multiple computers operating near or generally remote from each other and typically interacting across a communications network. Examples of communication networks include local area networks ("LANs") and wide area networks ("WANs"), the internet (e.g., the internet), networks including satellite links, and point-to-point networks (e.g., ad hoc point-to-point networks). The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

Fig. 10 illustrates an example computer system 1000 that includes a processor 1010, a memory 1020, a storage 1030, and an input/output device 1040. Each of the components 1010, 1020, 1030, and 1040 may be interconnected, for example, by a system bus 1050. Processor 1010 is capable of processing instructions for execution within system 1000. In some implementations, the processor 1010 is a single-threaded processor, a multi-threaded processor, or other type of processor. Processor 1010 is capable of processing instructions stored in memory 1020 or on storage 1030. Memory 1020 and storage 1030 may store information within system 1000.

Input/output device 1040 provides input/output operations for system 1000. In some embodiments, input/output device 1040 may include one or more of the following: network interface devices, such as ethernet cards; serial communication means, such as an RS-232 port; and/or wireless interface devices such as 802.11 cards, 3G wireless modems, 4G wireless modems, and the like. In some embodiments, the input/output devices may include driver devices configured to receive input data and send output data to other input/output devices, such as a keyboard, printer, and display device 1060. In some embodiments, mobile computing devices, mobile communication devices, and other devices may be used.

An exemplary method 4200 of detecting the presence of a target analyte in a fluid sample is shown in fig. 42.

In method 4200, a plurality of magnetic beads is provided to a first fluid sample (4202). The plurality of magnetic beads includes a first binding moiety that specifically binds to a target analyte.

The plurality of magnetic beads is allowed to bind to the target analyte within the first fluid sample (4204).

The magnetic beads are transferred from the first fluid sample to a second fluid sample (4206). The second fluid sample includes a second binding moiety that specifically binds to the target analyte, and the second binding moiety is bound to the active enzyme. Transferring the magnetic beads includes immersing the sheath in a first fluid sample, placing a magnet in the sheath immersed in the first fluid sample such that the magnetic beads adhere to the sheath, removing the sheath containing the magnet from the first fluid sample, and immersing the sheath containing the magnet in a second fluid sample.

Allowing a second binding moiety within the second fluid sample to bind to the target analyte bound to the first binding moiety of the magnetic beads (4208).

The second fluid sample comprising a plurality of magnetic beads and a second binding moiety is combined with a solution of an electron mediator to obtain a third fluid sample (4210).

A third fluid sample is provided to the sample detection zone (4212) of the substrate. The sample detection region is disposed on the first electrode.

The third fluid sample is exposed to a magnetic field to retain a plurality of magnetic beads in the third fluid sample proximate to the first electrode (4214). The first electrode is electrically coupled to a potentiostat.

A redox reaction between the electron mediator and the active enzyme is induced in the third fluid sample (4216).

The output of the potentiostat is monitored to determine the presence of the target analyte in the third fluid sample (4218). The output of the potentiostat is varied by redox reactions.

Method 4200 may be performed, at least in part, using one or more of the devices and kits described herein. In some cases, method 4200 may be performed to identify analytes, such as cells, extracellular vesicles such as microvesicles, membrane particles, apoptotic vesicles or exosomes (e.g., transmembrane and cytoplasmic proteins, mRNA, DNA and microrna), peptides, proteins, lipids, metabolites and other molecules, that are free-floating (e.g., in serum or solution) or expressed on the surface of biological structures (e.g., on the surface of extracellular vesicles or cells). In some cases, method 4200 can be used to provide more effective care to a patient (e.g., by enabling a caregiver to diagnose and/or administer treatment in a more informed manner). In some cases, method 4200 can be performed to provide insight into the composition of a substance (e.g., a food product).

Application example of the novel detector arrangement

The embodiments described herein can be used in a variety of different applications, including the detection and quantification of various peptides, proteins, lipid metabolites, and other molecules that are either free-floating (e.g., in serum or solution) or expressed on the surface of biological structures (e.g., on the surface of extracellular vesicles or cells). In some cases, the detection and quantification of molecules may provide insight into a particular biological or pathogenic process, a particular progression of a particular disease, or some other biological condition.

Examples of various applications are discussed in more detail below and in the examples section.

Extracellular vesicle screening-cancer diagnosis

For example, magnetic electrochemical sensing can be used to detect the presence of biomarkers of cancer on extracellular vesicles.

There is increasing evidence that extracellular vesicles (Ev) are an effective readout for cancer therapy. For example, exosomes have become effective biomarkers. Exosomes are nanoscale vesicles actively secreted by cells. These vesicles carry the molecular components of the cell from which they are derived, including transmembrane and cytoplasmic proteins, mRNA, DNA and microrna, and can therefore be used as cellular substitutes. In combination with their relative abundance and ubiquity in body fluids (e.g., serum, ascites, urine, cerebrospinal fluid), exosomes may provide unique advantages for longitudinal monitoring. Exosome analysis is minimally invasive and provides a relatively unbiased readout of the overall tumor burden, with little effect from sample scarcity or intratumoral heterogeneity.

Electrochemical sensing can be an effective detection means that is easy to apply in a clinical setting. Electrochemical sensing can achieve high sensitivity by amplifying the signal of the redox active reporter. As described herein, the sensing system can measure the current induced by the redox-active reporter and can be implemented as a compact and low power-consuming portable device.

Embodiments of the sensing system, as described herein, provide various benefits. For example, cell-specific exosomes can be isolated directly from complex media without extensive filtration or centrifugation. Furthermore, the assay can achieve high detection sensitivity by magnetic enrichment and enzymatic amplification. Furthermore, with an electrical detection scheme, the sensor can be miniaturized and expanded for parallel measurements.

For example, ovarian cancer exosomes are typically rich in CD63. Thus, embodiments of the sensing system can be used to analyze EV populations (e.g., exosomes) expressing CD63 as a means of diagnosing ovarian cancer.

For example, the magnetic beads may be conjugated with an antibody specific for CD63. These magnetic beads can be mixed with a biological sample (e.g. plasma) containing exosomes, so that CD 63-expressing exosomes can be magnetically captured. Accordingly, the sample may be treated with a second molecule specific for exosomes expressing CD63 (e.g., a labeled ligand such as a biotinylated antibody specific for CD 63) and treated with a third molecule specific for the second molecule and having an oxidase (e.g., streptavidin-HRP). The sample can then be mixed with an electron mediator solution (e.g., a solution containing 3,3', 5' -tetramethylbenzidine, TMB).

The sample can then be analyzed using the sensing system described herein. Since exosomes expressing CD63 have been captured by magnetic beads, they are concentrated near the electrodes of the sensing system. In addition, a redox reaction is induced between the electron mediator and the oxidase enzyme due to the potential induced across the electrodes (e.g., the working electrode and the reference electrode). As a result, a current is induced on one electrode (e.g., the counter electrode), which is associated with the presence and prevalence of exosomes expressing CD63. Accordingly, the current may be measured by the MCU or other computing device, and the resulting information may be used for investigation or diagnostic purposes. For example, a relatively high current may correspond to a relatively high concentration of exosomes expressing CD63, and may be indicative of ovarian cancer in the patient.

In some cases, the output of the potentiostat may be compared to a threshold or reference level, and the presence or absence of cancer in the patient may be diagnosed based on the comparison. For example, if the output of the potentiostat is sufficiently high (e.g., the current exceeds a reference or threshold level), a diagnosis may be made as to the presence of cancer. However, if the output of the potentiostat is relatively low (e.g., the current does not exceed a reference or threshold level), a diagnosis may be made as to the absence of cancer. In some cases, the instrument may automatically or semi-automatically perform diagnostics based on the measurements.

Although the detection of exosomes expressing CD63 in blood is described above, this is only one example. In practice, embodiments of the sensing system can be used to detect any biomarker (e.g., a biomarker associated with a different organism or pathogenic process, disease, or other biological condition) that is free floating (e.g., free floating in plasma, urine, or any other biological sample) or expressed on the surface of a biological structure (e.g., on the surface of an extracellular vesicle or cell). As an example, in some embodiments, the sensing system may be used to detect one or more of the following biomarkers: CD2, CD3, CD45, CD52, HLA-ABC, CD81, CXCL10 or CXCL9 or any other immune cell marker. As another example, in some embodiments, the sensing system can be used to detect biomarkers for one or more of: CD24, epCAM, CA125, EGFR, HER2, MUC1, CD44v6, CEA, mesothelin, trop2, GPC1, WNT2, grp94, SSTR2, EGFRv3, IDH1-R132, GPA33, KRAS, CD166, CD133, MET, B7H3, CD63, CD9, or CD81.

Extracellular vesicle screening-organ rejection test

As another example, magnetic electrochemical sensing can be used to detect transplant organ rejection in a patient.

For example, in patients experiencing transplant kidney rejection, CD 3-expressing EVs are often found in the urine of patients. Thus, embodiments of the sensing system can be used to analyze CD 3-expressing EV populations (e.g., exosomes) as a means of detecting transplant kidney rejection at an early stage of rejection.

For example, the magnetic beads may be conjugated with an antibody specific for CD 3. These magnetic beads can be mixed with a biological sample containing exosomes (e.g., urine) so that CD 3-expressing exosomes can be magnetically captured. Accordingly, the sample may be treated with a second molecule specific for CD 3-expressing exosomes (e.g., a labeled ligand such as a biotinylated antibody specific for CD 3) and treated with a third molecule specific for the second molecule and having an oxidase (e.g., streptavidin-HRP). The sample can then be mixed with a solution of an electron mediator (e.g., a solution containing 3,3', 5' -tetramethylbenzidine, TMB).

The sample can then be analyzed using the sensing system described herein, and the resulting information can be used for research or diagnostic purposes. For example, a relatively high current may correspond to a relatively high concentration of CD 3-expressing exosomes and may be indicative of renal rejection by the patient. In some cases, patients may be screened periodically after kidney transplantation, so that kidney rejection is detected quickly at an early stage. Since the monitoring method is non-invasive, the monitoring method can be repeated with minimal risk to the patient and in a cost-effective manner.

In some cases, the output of the potentiostat may be compared to a threshold or reference level, and a determination may be made as to whether the patient has rejected the organ transplant based on the comparison. For example, if the potentiostat output is high enough (e.g., the current exceeds a reference or threshold level), a conclusion may be made that the patient has rejected the organ. However, if the potentiostat output is relatively low (e.g., the current does not exceed a reference or threshold level), then a conclusion may be made that the patient has not rejected the organ. In some cases, the instrument may make a conclusion automatically or semi-automatically from the measurements.

Although the detection of exosomes expressing CD3 in urine is described above, this is merely an illustrative example. Embodiments of the sensing system, as described herein, can be used to detect any biomarker that is free-floating or expressed on the surface of a biological structure to detect rejection of transplanted kidneys or rejection of other transplanted organs.

Food testing

As another example, magnetic electrochemical sensing can be used to detect the presence of allergens in food products.

Over 5000 million americans have some kind of food reaction. In addition, even minute amounts of food antigens can trigger acute allergic reactions, a potentially life-threatening hypersensitivity reaction that requires the injection of epinephrine. Although the results of immunotherapy trials are encouraging, the main approach still relies on avoiding food. The "food allergen labeling and consumer protection act" (FALCPA) requires that the food label inform the consumer of the allergenic substances in the product. Even so, false labeling or cross-contamination in manufacturing still presents regulatory challenges. Furthermore, FALCPA only supervises packaging of food, and does not supervise restaurant-offered food. Food labeling outside the united states is less stringent and food allergies often affect travelers. Thus, the ability to rapidly test for common allergens in foods can bring significant benefits to the consumer.

As an example, a consumer may be provided with an assay kit having a magnetic electrochemical sensing system and a sample preparation system, such as the kits described in fig. 6-9 herein. A consumer may use the kit to prepare a food sample for examination, extract a food antigen of interest (e.g., an allergen to which the user may be allergic, such as wheat, peanut, hazelnut, milk, and egg white) from the sample using antigen-specific magnetic beads, and aggregate the antigen onto an electrode of a sensing system. The consumer can then analyze the sample using a sensing system (e.g., using a second molecule, a third molecule, and an electron-mediated solution) to determine the presence and/or prevalence of antigens in the food product so that they can make more informed decisions about their diet.

In some cases, the consumer may also be provided with a control application for a computing device (e.g., a smartphone) that enables the consumer to control the sensing system and/or view measurement information from the sensing system. For example, the control application can be used to customize the sensing application for a particular allergen (e.g., by calibrating the measurement of a particular allergen and its corresponding sample preparation process). As another example, the control application may be used to record measurements for future viewing. As another example, the control application may be used to track the origin of a particular food product so that the user may later revisit the particular location (e.g., obtain a food product that does not contain a particular allergen) or avoid the particular location in the future (e.g., avoid obtaining a food product that contains a particular allergen).

In some cases, the output of the potentiostat may be compared to a threshold or reference level and based on the comparison, it is determined whether a particular allergen is present in the food product. For example, if the potentiostat output is sufficiently high (e.g., the current exceeds a reference or threshold level), then a conclusion may be made that the food product contains a particular allergen (e.g., to a degree sufficient to elicit or likely to elicit an allergic reaction). However, if the potentiostat output is relatively low (e.g., the current does not exceed a reference or threshold level), then a conclusion may be made that the food product does not contain a particular allergen (e.g., to the extent that it does not cause or is unlikely to cause an allergic reaction). In some cases, the instrument may automatically or semi-automatically perform the assay based on the measurement.

Although exemplary allergens are described above, these are merely illustrative examples. In practice, embodiments of the sensing system can be used for the detection of any allergen using suitable allergen-specific magnetic beads.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims of the invention.

Example 1 cancer diagnosis

The purpose of this example is to demonstrate the use of magnetic electrochemical sensing to screen exosomes as biomarkers, such as CD63, associated with ovarian cancer progression.

Summary of the devices

A miniaturized magnetic electrochemical sensing system (integrated magneto-electrochemical exosome system, herein referred to as "iMEX") with eight independent channels was constructed (see fig. 11). Each channel is equipped with a potentiostat capable of measuring a wide range of currents (+ -7.5 mua). The sensor can measure the signals from eight electrodes simultaneously. Small cylindrical magnets are located below the electrodes to gather immunomagnetically captured exosomes.

The input signal is conditioned by a low pass filter (cut-off frequency, 5 Hz) to suppress high frequency noise. A circuit schematic of a low pass filter embedded in each potentiostat. Vw-set is connected to a digital-to-analog converter for potential control, vw is connected to the working electrode, and Vout is connected to an analog-to-digital converter for signal digitization (see fig. 12A). The cutoff frequency of the low-pass filter is set to 5Hz (Rf =300k Ω, cf =0.1 μ F). A comparison of the measurements between the iMEX sensor and the commercial equipment (SP 200, bio-Logic) is shown in FIG. 12B. A potential of 200mV (vs. Ag/AgCl reference electrode) was applied to a solution of 0.2mM ferrocyanide (in 0.1M KCl). The current levels measured using the iMEX sensor and commercial equipment showed good agreement. With a low pass filter, the noise level drops significantly.

Eight potentiostats are connected to the digital-to-analog converter for potential control, to the analog-to-digital converter for signal digitization, to the multiplexer for channel selection, and to the microcontroller unit for system operation (see fig. 4C and 13A-D). The sensor system has eight potentiostats, an 8. Each potentiostat has three electrodes: reference (R), pair (C) and work (W). As shown in fig. 13A, the system includes two potentiostats (AD 8608, analog device). The parallel circuit of R2 and C2 (or R4 and C4) forms a transimpedance amplifier with a low-pass filter with a cut-off frequency of 5 Hz. As shown in fig. 13B, a microcontroller unit (Arduino Nano, arduino) is used for serial communication with an external device through USB. As shown in fig. 13C, the system also includes a digital-to-analog converter unit (DAC 8552, texas Instruments). One output is connected to the analog-to-digital converter unit (DAC _ OUT 1) and the other to all potentiostats (DAC _ OUT 2). Each output port is connected to a low pass filter (L1, C15, C17, R20 for DAC _ OUT1 and L2, C19, C20, R22 for DAC _ OUT 2) to minimize noise. As shown in fig. 13D, the system also includes an 8-channel multiplexer (AD 0700, analog Devices) and an Analog-to-digital converter unit (ADC 161S626, texas Instruments). The multiplexer is controlled by the microcontroller unit through its three address ports (MUX _ A0-MUX _ A2).

We package the device as a hand-held device, as shown in fig. 14. The device has small and exquisite appearance (9 is multiplied by 6 is multiplied by 2 cm) ³ ). Card edge connectors are used to quickly connect electrode cartridges. A magnet holder containing 8 cylindrical magnets was placed under the electrode cartridge. These magnets are used to concentrate the magnetic beads on the sensor surface. By polling (polling) each channel (50 ms per channel), iMEX effectively provides simultaneous readings of all electrodes.

All data was monitored and analyzed by custom designed software. The iMEX sensor is controlled via a computer through a USB interface. The current generated by the electrochemical reaction is monitored on the selected channel. After 60 seconds, the average level of current is shown in the range of 40-45 seconds (see FIG. 15).

Assay protocol

Fig. 16 summarizes the iMEX assay protocol. Exosomes were first captured onto immunomagnetic beads. A secondary antibody with an oxidase (horseradish peroxidase; HRP) was then used, and the beads were then mixed with a chromogenic electron mediator (3, 3', 5' -tetramethylbenzidine; TMB) that generated a current when encountering HRP. The assay protocol is shown in more detail in figure 17. Magnetic beads conjugated with antibodies against CD63 were loaded into body fluid (or PBS) for exosome isolation. The captured exosomes are labeled with antibodies to a target protein marker (e.g., epCAM or HER 2). The antibody was conjugated to biotin. Streptavidin-conjugated HRP enzyme was mixed with the beads. Each step was followed by a one minute magnetic wash step. All measurements were performed at room temperature within 1 hour.

The use of magnetic beads significantly simplifies the assay procedure: excess reagents (e.g., antibodies, enzymes) are removed via magnetic washing and the captured exosomes are magnetically concentrated on the electrodes to increase detection sensitivity.

We applied the chronoamperometry for signal detection: the current generated by the reduction of TMB was monitored while a reduction potential (100 mV versus Ag/AgCl reference) was applied to the working electrode. 1 minute after application of reduction potentialThe internal current level (I) reached a plateau (see fig. 18). Current difference (Δ I) between CD 63-bead and IgG-bead samples _M ) Is used as a representative value of the target protein marker. Abs, antibodies. M, a marker. We averaged the current level (I) from 40 to 45 seconds as a representative value.

To capture the exosomes, we used magnetic beads coated with antibodies to the tetraspanin (transmembrane protein rich in exosomes). We first compared the signal levels of different sized beads (diameter, 2.7 μm and 8.8 μm). When the total surface area of the beads was matched to capture similar amounts of exosomes, the measured signal levels were almost the same (see fig. 19A). iMEX assays were performed using magnetic beads of different sizes (2.7 and 8.8 μm in diameter). The bead concentrations were 6X 10 respectively ⁷ Perml and 6X 10 ⁶ mL to provide the same capture area. Exosomes were collected from OV90 cell cultures and diluted in PBS at a concentration of 8 × 10 ⁸ and/mL. This result can be explained by the diffusivity in the porous medium: the effective diffusivity (De) of the stacked beads can be expressed as D _e ＝D ₀ ·ε ^m Wherein D is ₀ Is the diffusivity in the free medium and epsilon is the porosity of the structure. In the case of uniformly sized beads, both ε (≦ 0.47) and m (= 3/2) are independent of bead size; the iMEX signal is expected to remain unchanged. We chose to use 2.7 μm beads; larger beads tend to sediment, requiring frequent shaking of the sample. Magnetic enrichment resulted in a-72% increase in assay signal compared to the non-enrichment protocol (see figure 19B). iMEX assays were performed with and without magnetic enrichment. For magnetic enrichment, a permanent magnet in the shape of a small coin is placed on the back of the working electrode. All measurements were performed in duplicate and data are shown as mean ± Standard Deviation (SD).

We next tested three representative tetraspanin proteins (CD 63, CD9, CD 81) as targets; these markers are reported to be enriched in exosomes. We prepared 2.7 μm magnetic beads specific for each marker. When applied to exosomes from different cell lines, CD 63-based capture showed consistently high signals. Figure 20 shows a comparison of the signals of three tetraspanin markers (CD 63, CD9 and CD 81) in cancer exosomes. The signal from CD63 was much higher than that of other markers in exosomes collected from ovarian cancer cell lines (CaOV 3, OV90 and OVCAR 3). Therefore, we chose to use CD63 as a marker for exosome enrichment.

For each target marker (M), we prepared a pair of magnetic beads: one conjugated to an antibody directed against CD63 (CD 63-bead) and the other conjugated to an antibody directed against isotype matched IgG (IgG-bead). Mixing exosomes with each bead type, followed by labeling with antibodies to the target marker; the net signal difference Δ IM (= I) is then obtained _CD63+M -I _IgG+M (ii) a See fig. 18). We used Δ I _CD63 Estimating total exosome loading and normalizing metric ξ _M (＝ΔI _M /ΔI _CD63 ) Is defined as the expression level of the marker of interest (M). Note that this scaling will compensate for the change in the number of exosomes in the sample.

iMEX verification

We applied the developed iMEX procedure to analyze exosomes for transmembrane proteins. For this validation study, we harvested exosomes from cell cultures (OV 90, OVCA 420) by conventional methods and incorporated them into Phosphate Buffered Saline (PBS) solutions (-10) ⁹ exosome/mL). Samples were aliquoted and processed by iMEX and enzyme-linked immunosorbent assay (ELISA). Comparative analysis showed a high correlation between the two methods (see fig. 21 ² = 0.931) confirming analytical capability of iMEX. However, the iMEX assay is faster (1 hour) and consumes a smaller amount of sample (10. Mu.L) than the ELISA (5 hours, 100. Mu.L).

We further tested iMEX for the detection of exosomes in biological fluids. Cancer exosomes were collected from cell cultures (OV 90) and different amounts of exosomes were incorporated into undiluted human plasma. Titration experiment establishes 3X 10 ⁴ The detection Limit (LOD) of exosomes, the dynamic range spanning four orders of magnitude (see fig. 22). Similar measurements with ELISA require more than 10 ⁷ Individual exosomes were used for reliable detection. The use of matched controls (IgG-beads) is important to compensate for background signals arising from sample-dependent, non-specific exosome binding.Detection limit is 3 x 10 ⁴ (iMEX) and 3X 10 ⁷ (ELISA). All measurements were performed in triplicate and data are shown as mean ± SD.

Analysis of protein markers in cell-derived exosomes

We applied iMEX to screen for exosome surface markers from a panel of ovarian cancer cell lines. Because iMEX enriches CD 63-positive (CD 63 +) exosomes and labels them as target proteins, we were able to examine how CD63+ exosomes closely reflect their originating cells. We selected six representative surface markers from previous studies: epithelial cell adhesion molecule (EpCAM), CD24, cancer antigen 125 (CA 125), human epidermal growth factor receptor 2 (HER 2), mucin 18 (MUC 18), and epidermal growth factor receptor 2 (EGFR). Measuring the cellular expression levels of these markers by flow cytometry; exosomes were harvested from conditioned cell culture media and analyzed with iMEX. Molecular analysis of cells and CD63+ exosomes was highly correlated (see fig. 23 showing Mean Fluorescence Intensity (MFI), darker areas indicating higher MFI, lighter areas indicating lower MFI), supporting the use of exosomes as cell substitutes. Four ovarian cancer cell lines (CaOV 3, OV90, OVCAR3 and OVCA 420) and one normal cell line (TIOSE 6) were screened for six putative cancer markers (via flow cytometry, fig. 23, left panel). Cell-derived exosomes were immunomagnetically captured (CD 63-specific) and assayed by iMEX (fig. 23, right panel). The analytical data showed a good match between the cells and the CD63 positive exosomes. iMEX assays were performed in duplicate and mean values are shown.

Clinically: direct analysis of plasma from ovarian cancer patients

The iMEX assay separates EV directly from plasma or serum and allows analysis in a rapid, high throughput manner-this is critical for successful integration into clinical workflow. To demonstrate clinical feasibility, we customized the iMEX assay for EV detection of ovarian cancer in blood (see fig. 24). Clinical plasma samples were aliquoted without any purification, and each aliquot (10 μ Ι _ per marker) was incubated with magnetic beads for EV capture (15 min) followed by magnetic washing. The bead bound EV was labeled sequentially with target marker (15 min) and HRP (15 min) and loaded onto the device. With 8 electrodes operating independently, we were able to measure four different markers (CD 63, epCAM, CD24 and CA 125) and their respective IgG-controls simultaneously. IgG-controls are useful for specific detection of target molecules in unpurified clinical samples. The entire assay was completed in 1 hour without the need for filtration and centrifugation.

We tested single time point plasma samples from 11 ovarian cancer patients and 5 healthy controls. EpCAM and CD24 expression levels in ovarian cancer patients were much higher than healthy controls and both indices showed high correlation (R) ² = 0.870) (see fig. 25 and 26). We next examined the potential of the iMEX serial EV test by measuring EpCAM and CD24 in plasma collected at two time points (2 months apart) from four ovarian cancer patients receiving drug treatment. iMEX assays were performed blindly to treatment response. For "non-responsive" patients, the expression levels of EpCAM and CD24 were increased, while "responsive" patients showed a significant decrease in both markers (see fig. 27). For non-responders, CD24 levels showed a more dramatic increase than EpCAM. All measurements were performed in duplicate. U., arbitrary units.

Fabrication of iMEX System

The device includes a microcontroller (Atmega 328, atmel Corporation), a digital-to-Analog converter (DAC 8552, texas Instruments), an Analog-to-digital converter (ADC 161S626, texas Instruments), a multiplexer (ADG 708, analog Devices) and eight potentiostats. Each potentiostat comprises two operational amplifiers (AD 8606, analog Devices): one amplifier maintains a potential difference between the working electrode and the reference electrode, and the other acts as a transimpedance amplifier to convert the current into a voltage signal. The current measurement range of the transimpedance amplifier is ± 7.5 μ Α. Eight-channel electrodes are commercially available (DropSens, spain).

Preparation of immunomagnetic beads

5mg of epoxy-coated magnetic beads (Dynabeads M-270epoxy, invitrogen) were suspended in 1mL of 0.1M sodium phosphate solution at room temperature for 10 min. The magnetic beads were separated from the solution with a permanent magnet and resuspended in 100. Mu.L of the same solution. Add 100. Mu.g of antibody against CD63 (Ancell) or the corresponding IgG (Ancell) and mix well. 100 μ L of 3M ammonium sulfate solution was added and the whole mixture was incubated at 4 ℃ overnight with slow inclined rotation. The beads were washed twice with Phosphate Buffered Saline (PBS) solution and finally resuspended in 2mL PBS containing 1% Bovine Serum Albumin (BSA). More details can be found in the manual provided by the magnetic bead manufacturer.

Biotinylation of labeled antibodies

A solution of 10mM N-hydroxysulfosuccinic biotin (Pierce) in PBS was incubated with the antibody for 2 hours at room temperature. Unreacted N-hydroxysulfo succinyl biotin was removed using a Zeba desalting spin column, 7K MWCO (Thermo Scientific). The antibody was kept at 4 ℃ until use.

iMEX assay

mu.L of exosome-spiked PBS solution (or plasma) was mixed with 50. Mu.L of immunomagnetic bead solution at room temperature for 15 minutes. The bead concentration was determined according to the following criteria: [ C ] _b ×V _b ×4πR _b ² ]/[C _e ×V _e ×πR _e ² ]>100, wherein C _b And C _e Bead and exosome concentrations, respectively; v _b And V _e The volume of the bead solution and the exosome-incorporated solution (or plasma), respectively; r is _b And R _e The average radius of the beads and exosomes, respectively. This requirement ensures that sufficient bead surface is available for exosome capture. Under our experimental conditions, R _e ～50nm，R _b ＝1.4μm，C _e ～10 ¹⁰ and/mL. Therefore, we adjusted the bead concentration to 10 ⁸ The volume is/mL. The magnetic beads were separated from the solution with a permanent magnet and resuspended in 80. Mu.L PBS (1% BSA). After vortexing for 5 seconds, the beads were separated and resuspended in 80. Mu.L PBS (1% BSA). mu.L of the antibody of interest (20. Mu.g/mL in PBS) was mixed with the beads for 15 minutes at room temperature. The magnetic beads were isolated and washed as before and resuspended in 50. Mu.L PBS (1% BSA). mu.L of streptavidin-conjugated HRP enzyme (diluted in PBS at 1. The magnetic beads were isolated and washed as before and resuspended in 7 μ L PBS. Mixing the prepared bead solutionAnd 20. Mu.L of UltraTMB solution (ThermoFisher Scientific) was loaded on top of the screen printed electrode. After 3 minutes, chronoamperometric measurements were started with the electrochemical sensor. The current levels were averaged over a range of 40-45 seconds.

Enzyme-linked immunosorbent assay (ELISA)

The CD63 antibody (Ancell) and IgG1 antibody (Ancell) were diluted to a concentration of 5. Mu.g/mL in PBS and added to a Maxisorp 96 well plate (Nunc) and incubated overnight at 4 ℃. After washing with PBS, the PBS blocking solution containing 2% BSA was added to the plate and incubated at room temperature for 1 hour. Then, will contain 10 ⁸ 100 μ L of PBS from each exosome was added to each well and incubated for 1 hour at room temperature. After the blocking solution was discarded, antibodies (1. Mu.g/mL) against each marker were added to each well and incubated at room temperature for 1 hour. Unbound antibody was washed three times with PBS. streptavidin-HRP molecules were added to each well for 1 hour at room temperature. After elution with PBS, the chemiluminescent signal was measured.

Flow cytometer

Each antibody 5X 10 ⁵ Individual cells were used for flow cytometry experiments. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min, then washed with PBS (0.5% BSA). Subsequently, cells were blocked with BSA (0.5% in PBS) and then incubated with primary antibody (4. Mu.g/mL). After primary antibody incubation, cells were washed, incubated with fluorophore-conjugated secondary antibody (2. Mu.g/mL; abcam) and washed. Fluorescence signals from the labeled cells were measured using a BD LSRII flow cytometer (BD Biosciences). The Mean Fluorescence Intensity (MFI) recorded was calculated using the following formula [ (Signal-IgG isotype control)/Secondary]And (6) normalizing. Blocking with antibody (primary and secondary) and incubation were performed at room temperature for 30 minutes each. Each washing step included 3 washes with PBS (0.5% BSA) at 300g for 5 minutes.

Cell culture

OV90, OVCAR3, OCVA420 and TIOSE6 cells were grown in RPMI-1640 medium (Cellgro). CaOV3 was cultured in Dulbecco's modified essential Medium (DMEM, cellgro). All media were supplemented with 10% FBS and penicillin-streptomycin (Cellgro). All cell lines were tested and without Mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, lonza, LT 07-418).

Isolation of exosomes from cultured cells

We harvested exosomes from the cell culture medium using conventional methods. The cells from passage 1-15 were cultured in vesicle-depleted medium (5% depleted FBS) for 48 hours. About 10 collections from conditioned media ⁷ The cells were centrifuged at 300g for 5 minutes. The supernatant was filtered through a 0.2- μm membrane filter (Millipore) and concentrated at 100,000g for 1 hour. After removing the supernatant, the exosome pellet was washed with PBS and centrifuged at 100,000g for 1 hour. The exosome pellet was resuspended in PBS.

Clinical sample preparation

The study was approved by the institutional review board of Dana-Farber/Harvard Cancer Center, following procedures in accordance with the institutional guidelines. Informed consent was obtained from all subjects (n = 11). Peripheral blood (15 mL) was removed from patients with ovarian cancer and centrifuged at 400g for 15 minutes to separate plasma from red blood cells and buffy coat. For each surface marker analysis 10 μ L plasma was used.

Conclusion

This example demonstrates the use of magnetic electrochemical sensing to screen exosomes for biomarkers associated with the progression of ovarian cancer, such as CD63.

One advantageous feature of the sensing system is the integration of vesicle separation and detection into a single platform. The use of magnetic actuation simplifies vesicle separation and subsequent assay steps, and electrochemical sensing facilitates high throughput screening and sensor miniaturization. Current research verifies these concepts: i) A portable detection system with parallel measurement capability is realized; ii) the system enriches exosomes directly from blood and analyzes their molecular information; iii) The whole system assay (i.e. exosome separation, labeling, detection) was completed within 1 hour, while only 10 μ Ι _ of plasma was consumed per marker. We also demonstrated the clinical potential of this system by analyzing EV in blood collected from ovarian cancer patients.

Bead-based magnetic enrichment brings several advantages in sensing systems. First, the method provides a convenient way of concentrating the signal source on the electrodes, which improves the detection sensitivity. Secondly, compared to surface-based capture where the antibody is immobilized on the chip surface, the bead-based approach is suitable for reliable and simpler conjugation chemistry and benefits from faster binding kinetics between the antibody and the exosomes. Third, the bead-bound vesicles can be easily recovered for downstream molecular analysis in tandem with a sensing system. For example, the beads-bound EVs can be eluted or lysed to analyze their nucleic acid content.

In this example, we focused on analyzing the CD63+ EV population (exosomes), which is driven by two factors: i) The signal from the CD63 capture was the highest of the four transmembrane protein markers tested; and ii) we and others have previously shown that ovarian cancer exosomes are rich in CD63. Analysis of this system found a high correlation in protein expression between CD 63-positive exosomes and their parental cells; this result demonstrates the potential use of CD 63-positive exosomes as cell substitutes. However, we note that the exosome-capture strategy can be extended to take into account the different EV types (e.g. CD63 negative) that may be present in the patient sample. Examining these populations may yield more accurate information to capture tumor heterogeneity. The sensing method can be easily used for this purpose by changing the capture antibody.

We envision a number of directions that further advance the technology. First, assay throughput can be increased by increasing the number of detection sites. Electrochemical sensing is ideally suited for such amplification: the sensing elements (electrodes) can be easily microfabricated into a large array format, and the signals (currents) can be read out by compact electronics with high-speed multiplexing. Secondly, the detection sensitivity can be improved by exploring a new design for electrochemical signal detection. The signal level is related to the surface area of the sensor and the amount of enzyme bound to the target entity; thus, higher sensitivity can be achieved by using nanostructured sensor surfaces or multi-labeled nanoparticles. Third, the detection target can be extended to include other exosome components. For example, exosomes carry various nucleic acids (e.g., mRNA, microrna); analysis of nucleic acids and exosome proteins will provide a more accurate snapshot of tumor status. Electrochemical sensing has been applied to detect trace amounts of nucleic acids (< 1 pM) without PCR amplification. It is expected that similar methods can be used to analyze exosome nucleic acids. The iMEX thus produced can be a powerful clinical tool for economical, scalable and comprehensive exosome analysis, thus deepening our understanding of tumor biology and accelerating effective cancer management.

Example 2 organ rejection test

The purpose of this example is to demonstrate the use of magnetic electrochemical sensing to screen exosomes for biomarkers associated with transplanted kidney rejection.

Cell culture

OV90, OVCAR3, OVCA420 and TIOSE6 cells were grown in RPMI-1640 medium (Cellgro). CaOV3 was cultured in Dulbecco's modified essential medium (Cellgro). All media were supplemented with 10% Fetal Bovine Serum (FBS) and penicillin-streptomycin (Cellgro). All cell lines were tested and there was no Mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, lonza, LT 07-418).

Isolation of EV from cultured cells

We harvested EV from the cell culture medium using conventional methods. The cells from passage 1-15 were cultured in vesicle-depleted medium (containing 5% depleted FBS) for 48 hours. About 10 collections from conditioned media ⁷ Cells were centrifuged at 300 Xg for 5 min. The supernatant was filtered through a 0.2 μm membrane filter (Millipore) and concentrated at 100,000 Xg for 1 hour. After removing the supernatant, the EV pellet was washed with PBS and centrifuged at 100,000 × g for 1 hour. EV pellets were resuspended in PBS.

Biotinylation of labeled antibodies

A solution of N-hydroxysulfosuccinic biotin (10mM, pierce) in PBS was incubated with the antibody at room temperature for 2 hours. Unreacted N-hydroxysulfo succinyl biotin was removed using a Zeba desalting spin column, 7K MWCO (Thermo Scientific). The antibody was kept at 4 ℃ until use.

Enzyme-linked immunosorbent assay (ELISA)

The CD63 antibody and IgG1 antibody (Ancell) were fractionatedSeparately diluted to a concentration of 5. Mu.g/mL in PBS and added to a Maxisorp 96 well plate (Nunc) and incubated overnight at 4 ℃. After washing with PBS, the PBS blocking solution containing 2% BSA was added to the plate and incubated at room temperature for 1 hour. Then, will contain 10 ⁸ 100 μ L of PBS from each exosome was added to each well and incubated for 1 hour at room temperature. After the blocking solution was discarded, antibodies (1. Mu.g/mL) against each marker were added to each well and incubated at room temperature for 1 hour. Unbound antibody was washed three times with PBS. Streptavidin-horseradish peroxidase (HRP) molecules were added to each well for 1 hour at room temperature. After elution with PBS, the chemiluminescent signal was measured.

iMEX assay

mu.L of exosome-doped PBS solution was mixed with 50. Mu.L of immunomagnetic bead solution at room temperature for 30 min. The bead concentration was determined according to the following criteria: [ C ] _b ×V _b ×4πR _b ² ]/[C _e ×V _e ×πR _e ² ]>100, wherein C _b And C _e Bead and exosome concentrations, respectively; v _b And V _e Volumes of bead solution and spiked exosome solution, respectively; r _b And R _e The average radius of the beads and exosomes, respectively. This requirement ensures that sufficient bead surface is available for exosome capture. Under our experimental conditions, R _e ～50nm，R _b =1.4 μm, and C _e ～10 ¹⁰ and/mL. Therefore, we adjusted the bead concentration to 10 ⁸ and/mL. The magnetic beads were separated from the solution with a permanent magnet and resuspended in 80. Mu.L PBS (1% BSA). After vortexing for 5 seconds, the beads were separated and resuspended in 80. Mu.L PBS (1% BSA). mu.L of the antibody of interest (20. Mu.g/mL in PBS) was mixed with the beads for 30 minutes at room temperature. The magnetic beads were isolated and washed as before and resuspended in 50. Mu.L PBS (1% BSA). mu.L of streptavidin-conjugated HRP enzyme (diluted in PBS with 1. The magnetic beads were isolated and washed as before and resuspended in 7. Mu.L of PBS. The prepared bead solution and 20. Mu.L of UltraTMB solution (ThermoFisher Scientific) were loaded on top of the screen printed electrode. After 3 minutes, transfer by electrochemistryThe sensor begins a chronoamperometric measurement. The current levels were averaged over a range of 40-45 seconds.

Isolation of EV from clinical samples

The study performed was approved by the institutional review board of the research institution, following procedures consistent with institutional guidelines. Informed consent was obtained from all subjects (n = xx). Urine samples (15 mL) were collected from kidney transplant patients and centrifuged at 400g for 15 minutes to separate plasma from red blood cells and buffy coat. For each surface marker analysis 10 μ L plasma was used.

Fig. 28 is a schematic of T cell-derived EV secretory canaliculi in the kidney. FIG. 29 shows histology of biopsy samples from renal transplant rejection patients. Fig. 30 shows a schematic of a 96-well plate format iMEX system for detection of Jurkat T cell-derived exosomes. The sensor can measure the signals of 96 samples simultaneously. Scanning electron microscopy revealed EV captured by CD3 antibody functionalized magnetic beads. A schematic of the iMEX assay is also shown. EV from jurkat cells or patient urine is captured by anti-CD 3 antibody conjugated magnetic beads. Subsequent biotinylated anti-CD 63 antibody and HRP enzyme labeling generates an electrochemical signal.

Fig. 31A shows the measured currents when detecting EVs with CD3 expression with the iMEX assay in the discovery group. Figure 31B shows ROC curves for determining sensitivity, specificity and accuracy of CD3 markers in the discovery group.

Fig. 32A shows the measured current when detecting EVs with CD3 expression with the iMEX assay in the validation set. Figure 32B shows ROC curves for determining sensitivity, specificity and accuracy of CD3 markers in the validation set.

Conclusion

This example demonstrates the use of magnetic electrochemical sensing to screen exosomes for biomarkers associated with transplanted kidney rejection as a means to detect rejection at its early stages.

Example 3 food testing

The purpose of this example is to demonstrate the use of magnetic electrochemical sensing to detect the presence of allergens in food products.

Adverse food reactions, including food allergies, food sensitivity and autoimmune reactions (such as celiac disease) affect 5-15% of the population and are still a considerable public health problem, requiring strict food avoidance and also the availability of epinephrine for emergencies. Given today's dependence on prepared foods and on meals outside, avoiding problematic foods is easy to do. We developed a portable, ready-to-use technology for rapid exogenous food antigen testing (referred to herein as "iEAT"). The system includes a disposable antigen retrieval device coupled with an electronic key fob reader for rapid sensing and communication. We optimized the prototype ifeat system to detect five major food antigens in peanuts, hazelnuts, wheat, milk and eggs. The time required for antigen extraction and detection using the iEAT is less than 10 minutes, and the detection sensitivity is below 0.003ppm, well below regulatory limits. When tested under restaurant conditions, we were able to detect cryptic food antigens, such as gluten in "gluten free" foods. The small size and rapid, simple testing of the ifeat system not only aids consumers, but also clinicians, food industries and regulatory agencies in improving food safety

Over 5000 million americans have some kind of food reaction. Food allergies cost $ 250 billion per year in the united states alone. Even minute amounts of food antigens cause acute allergic reactions, a potentially life-threatening hypersensitivity reaction, and require adrenaline injection. Although the results of immunotherapy trials are encouraging, the primary approach still relies on avoiding food. The "food allergen identification and consumer protection act" (FALCPA) requires that the food identification informs the consumer about the allergenic substance in the product. Even so, false identifications or cross-contamination in manufacturing still present regulatory challenges. Furthermore, FALCPA only supervises packaged food, not restaurant-provided food. Food labeling outside the united states is less stringent and food allergies often affect travelers. Therefore, the ability to rapidly test for common allergens in foods is a major unmet need.

The system includes a disposable allergen extraction device and an electronic key fob reader for sensing and communication. The extraction kit captures and aggregates food antigens from dispersed food. The captured allergens are then quantified using a miniaturized key fob reader. In summary, the iet system enables quantitative allergen detection in a short and operable time frame (i.e. <10 minutes for the entire assay). We have specifically designed ifeat to facilitate consumer-based operations: i) The extraction kit is simple to use, low in price and disposable; ii) the detection is fast, reliable and accurate; and iii) an embedded communication protocol allows users to record and upload information to the cloud server using time and place tags. We optimized the eae prototype to detect five representative allergens from wheat, peanut, hazelnut, milk and egg white. The rapid ifeat assay achieves high sensitivity, far exceeding the ELISA of the gold standard. We also show the practical use of ifeat to measure these allergens in common foods.

iEAT analysis

Fig. 33A depicts a portable ifeat system that includes a key fob reader, an extraction kit, and a smartphone App, as described in fig. 6-9 herein. The system comprises a key fob size detector, an electrode chip, and a disposable cartridge for allergen extraction. The detector is connected with the smart phone and used for controlling the system and uploading data to the cloud server.

The first step in the sensing is the extraction of the allergen via the use of a specially designed disposable kit enriched by immunomagnetism (see fig. 33B). The allergen is captured on magnetic beads and labeled with a secondary antibody conjugated to an oxidase (horseradish peroxidase, HRP). Disposable kits have been developed to process samples; the encapsulated magnet was collected and the MB was redispersed. When mixed with a chromogenic electron mediator (3, 3', 5' -tetramethylbenzidine, TMB), the beads generate an electrical current due to the oxidation of the TMB. The current is then measured through the electrodes. The signal was amplified by enzymatic reaction and magnetic enrichment of MB. The electrical detection scheme allows quantitative measurements to be made using miniaturized electronics. Furthermore, the use of magnetic beads as solid substrates improves the assay performance in two ways.

First, the extraction process and sample handling is simplified via magnetic actuation. To enable portable operation, we have designed a simple encapsulated magnetic strip for bead collection and resuspension (see fig. 8), which eliminates the need for special equipment (e.g., centrifuges, pipettes). The extraction kit simplifies the allergen extraction and labeling process. A magnet was attached to the end of the glass rod and enclosed with a quartz tube. The assembly was inserted into a sample tube containing a mixture of food extract and immunomagnetic beads. The collected beads were easily transferred to different tubes for washing and labeling.

Second, the electrochemical signal is amplified by magnetically aggregating the beads on top of the electrodes. For this purpose, we have designed an electrode holder with a small magnet (see fig. 6D). The support has a small magnet, one for each electrode, for focusing the magnetic beads. Showing a holder for a single electrode.

We have designed the iet reader as a key-fob sized device for portability (see fig. 6A-6C). The mini-reader not only detects and displays the results, but also wirelessly communicates with the smartphone via bluetooth, transmitting the test results and other information to the cloud server for Web-based data collection and sharing among users. The smartphone application communicates with the ifeat device via bluetooth and uploads the data to the cloud server. The functions include: (i) taking a picture of the user and analyzing the food; (ii) setting up a detection channel (e.g., allergen type); (iii) displaying the measurement; (iv) tracking food intake; and (v) storing the measurement time and the location on the map.

In addition, the communication capability provides an expanded user interface for system control, data storage, and wireless battery charging. The device has a number of components including a potentiostat for current measurement, a microcontroller unit (MCU) for signal processing, a mini-display screen, a rechargeable battery, and a card edge connector for plugging in an electrode plate. The miniaturized device is a stand-alone unit that measures the current and displays the allergen concentration according to a pre-loaded look-up table.

As shown in fig. 34, the iet reader includes a custom designed potentiostat connected to a digital-to-analog converter for potential control and an analog-to-digital converter for signal digitization. The MCU is programmed to measure the current from the working electrode (W) to the counter electrode (C) while maintaining a constant potential between the working and reference (R) electrodes. The circuit is designed to accommodate a single electrode or an array of electrodes. For a single electrode, electrical contact occurs at the top side of the card edge connector; for a multi-channel array, contact occurs through the bottom side of the card edge connector. The ifeat reader automatically senses the mode of operation (i.e., single or multi-channel). For multi-channel detection, the MCU polls the electrodes sequentially through the multiplexer.

We used potassium ferrocyanide [ K ] ₄ Fe(CN) ₆ ]The standard was benchmark tested for iEAT performance with a commercial bench-top potentiostat (SP-200, bio-Logic). We have generated K for each system ₄ Fe(CN) ₆ And (6) calibrating the curve. Next, we measured different K ₄ Fe(CN) ₆ Test samples at concentration, and we obtained their concentration from the calibration curve. We observed a good match between the two systems (R) ² =0.995; see fig. 35). The iet reader also showed good accuracy: coefficient of Variation (CV) of five replicate measurements<<xnotran> 4.1%, CV (</xnotran><4.9%) were equivalent. However, the physical dimensions of the iEAT reader (5.5X 2.5X 2.4 cm) ³ 35 g) than desk top system (38X 21X 17 cm) ³ 6 kg) is much smaller and 8 parallel measurements can be made.

Antigen extraction

We first optimized the antigen extraction protocol. Our goal is to minimize extraction time and cost while maximizing recovery. We used five major protein antigens as extraction targets: gliadin (wheat), ara h1 (peanut), cor a1 (hazelnut), casein (milk) and ovalbumin (egg white). Simulated food was prepared by incorporating a known amount (10 ppm) of protein into white rice. We tested three extraction buffers: 2-mercaptoethanol (2-ME), tris- (2-carboxyethyl) phosphine (TECP/GUA) enhanced with guanidine and TECP with N-lauroylsarcosine (TECP/sarcosyl). 2-ME reduction buffers are commonly used to extract proteins from highly processed foods, but have a strong odor. We prepared TECP-based reductants as potential user-friendly alternatives.

For a given extraction buffer, we varied the incubation time and temperature, and we monitored the recovery. FIG. 36A shows an example of extraction of Ara h1 with 2-ME buffer. Extraction is effective even at room temperature; more than 60% of the antigen was recovered within 2 minutes of incubation. The extraction rate increases with increasing temperature. For example, after heating in a microwave oven (1100W) for 20 seconds, incubation in extraction buffer for 1 minute increased the yield to 80%. All three extraction buffers showed similar performance for the five tested antigens (see fig. 36B and 36C). Therefore, we chose to use a tasteless, low-cost TECP/sarcosyl extraction buffer (see Table 1)

TABLE 1 iEAT detection limits for five food antigens

Allergens	Detection limit (ppm)	Action threshold 1 (ppm).)
			Gliadins	0.076	20
Ara h1	0.007	8
			Cor a1	0.090	10
Casein protein	0.812	50
			Egg white protein	0.003	20

* VITAL (volume incorporated traffic alert laboratory) created a series of action levels. Action threshold 1 does not require the food manufacturer to label or declare.

We also constructed a small heating device (see fig. 37) to speed up the extraction process. The extraction conditions were set to incubate at-60 ℃ for 2 minutes.

Measuring

To capture the predetermined antigen, we prepared immunomagnetic beads (2.8 μm diameter) by conjugating monoclonal antibodies to the beads. Control beads were conjugated to isotype matched IgG antibodies. The optimal bead concentration using peanut allergen titration assay was 8X 10 ⁶ beads/mL (see FIG. 38A). Magnetic beads specific for peanut allergen (Ara h 1) were used to detect 20ppm Ara h1. Concentration of beads>10 ⁷ mL ^-1 Resulting in signal saturation. The optimum bead concentration was set at 8X 10 ⁶ mL ^-1 . Similar experiments were repeated for other allergens. Data shown are mean ± standard deviation from duplicate measurements.

After incubation with food extract at room temperature (3 min), the beads were collected with an envelope magnet and transferred to fresh buffer for washing. Subsequently, the beads were labeled with HRP-conjugated antibody (3 minutes at room temperature), washed and mixed with TMB to generate a signal. The current stabilized within 60 seconds after the application of the reduction potential (-0.1V) (see fig. 38B, dynamic current response). The measured signals are averaged between 50 and 60 seconds. The net current difference between the background and the target sample was used as an analytical measurement. Thus, we programmed the iet reader to average current levels between 50 and 60 seconds. The current level from the control beads was about-110 nA in the different allergens, and the difference in current between the target sample and the background was defined as the net signal. Total assay time including allergen extraction was <10 min.

To facilitate storage and transport of reagents, we further lyophilized the immunomagnetic beads and antibodies. We tested different lyophilization media (PBS, sucrose) and storage conditions (refrigerated, room temperature). No significant difference in the activity of the agent exists; all lyophilized reagents retained their activity (> 96%) after four weeks of storage (see fig. 39A and 39B). Referring to fig. 39B, the immunomagnetic beads and the detection antibody are lyophilized in PBS or sucrose. The lyophilized product was stored at room temperature or 4 ℃ and used to detect 10ppm peanut allergen. The agent retains its activity regardless of excipient type and storage conditions. All measurements were repeated and data are shown as mean ± standard deviation. In view of its ready availability, we chose PBS as excipient, and for ease of use, we chose ambient conditions for storage of lyophilized reagents.

Analysis of Performance

We next generated response curves by varying the target allergen dose (see fig. 40A for Ara h1, and fig. 40B for others); these curves are loaded into the iEAT reader as a lookup table. The iet assay is highly sensitive, accurate, allowing for robust allergen quantification. Limit of detection (LOD), defined as 3 σ · m ^-1 (where σ and m are the standard deviation and the slope of the calibration curve, respectively), which is more than 220 times lower than the priming dose (ED) threshold (see Table 1). Intra-assay variation estimated by measuring three different concentrations of standard (1, 5 and 10 ppm) with six replicates<5% (see FIG. 40C), and the inter-assay differences were determined<5 percent. For comparison, we also tested the same samples by ELISA. The iEAT results correlated well with the ELISA measurements (see figure 40d ² = 0.995). However, the iet assay is much faster (10 minutes compared to 2 hours for ELISA). To test the specificity of the assay, we applied the target probe to different allergen standards (5 ppm). As shown in fig. 40E, the specific signal was 20-fold greater than the non-target sample.

In situ testing

We next tested the consumer food using the iEAT platform. We first tested a set of packaged staple foods (bread, milk, cereals) and desserts (biscuits, ice cream). Small portions (about 1 g) of food were treated in 2 minutes as described above and the extracts were assayed for gliadin, ara h1 (peanut), cor a1 (hazelnut), casein (milk) and ovalbumin (egg white). The results of the analysis are summarized in fig. 41A. As expected, a product with a particular signature (e.g., "gluten free", "nut free") is essentially free of the listed allergens. However, most products contain at least one unspecified antigen; for example, a nut-free biscuit brand contains gluten, whereas a gluten-free brand contains peanut allergens.

We next measured the food obtained from the restaurant (hamburger, sauce salad, pizza and beer). The analysis results (see fig. 41B) show the expected allergens, such as gluten in hamburgers and pizzas, but we also detected unexpected antigens from food processing. For example, salad contains gluten, which may be from salad dressing. We have also found ovalbumin and casein in beer, which is not surprising as egg white is used to improve the foam properties and casein is used to stabilise the beer during brewing.

Using the interface of iEAT with smartphone, we tracked individual dietary intake, and recorded antigen data with time points in the cloud server (see fig. 41C). As an example, we investigated gluten-free menu items from seven current-location restaurants and recorded the results with regional information (e.g., food name, gluten content). In the "gluten free" commercial, we observed broad spectrum antigen levels (1 ppm to >100 ppm); gluten for three commercial products far exceeded regulatory limits (see figure 41D, left). These results are then used to create an evidence-based restaurant map (see fig. 41D, right). These maps can be shared and expanded to include other antigens for personalization.

Material

The following chemical and biochemical reagents were used as received: superparamagnetic beads (6.7X 10) ⁷ The ratio of the beads to the mg of the beads,

m-270epoxy, invitrogen); bovine serum albumin (98% or more, BSA, sigma); ovalbumin (OVA, invivoGen), gliadin from wheat (Sigma); casein from milk (Sigma); sulfuric acid (1N, fluka);

high sensitivity streptavidin-horse radish peroxidase (strep-HRP, thermo Scientific); monoclonal mouse IgG ₁ (1.0 mg/mL, ancell); monoclonal mouse IgG _2a (1.0 mg/mL, ancell); gliadin peptide antibody (14D5, 1.0mg/mL, monoclonal mouse IgG) _2a Enzo Life Sciences); anti-gliadin antibody (15.5 mg/mL, HRP conjugated, polyclonal rabbit IgG, abcam; HRP conjugated to gliadin ELISA kit from MIoBS); ovalbumin antibody (6C8, 0.98mg/mL, monoclonal mouse IgG ₁ Thermo Fisher); anti-OVA polyclonal antibody (HRP conjugated, ovalbumin ELISA kit from MIoBS); a naturally purified Arachis hypogaea allergen (peanut protein Ara h1, indor Biotechnologies); anti-Ara h1 antibody (2C12, 2.7mg/mL, monoclonal mouse IgG ₁ Indoor Biotechnologies); biotinylated anti-Ara h1 antibody (2F 7, monoclonal mouse IgG) ₁ Indoor Biotechnologies); peanut standards (Diagnostic Automation, inc.), rabbit anti-bovine casein polyclonal antibody (2 mg/mL, AGRO-BIO); anti-casein polyclonal antibody (HRP conjugated, casein ELISA kit from MIoBS); hazelnut protein standard substance (A)

Hazelnut ELISA kit and Diagnostic Automation, inc.); rabbit polyclonal antiserum against hazelnut protein (anti-hazelnut antibody, accurate Chemical)&Scientific Corporation); HRP conjugated anti-hazelnut antibody (c) ((r))

Hazelnut ELISA kit); sucrose (99.5% or more, sigma); 1-step super 3,3', 5' -Tetramethylbenzidine (TMB) ELISA substrate solution (Thermo Scientific); tris (hydroxymethyl) aminomethaneAlkane hydrochloride (Tris-HCl, 99% or more, sigma); 2-mercaptoethanol (2-ME, ≧ 99%, aldrich); guanidine hydrochloride (GUA, ≧ 99%, sigma); sodium dodecyl sulfate (SDS, ≧ 99%, sigma-Aldrich); n-lauroylsarcosine (. Gtoreq.95%, sigma); tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 98% or more, aldrich); tween 20 (Sigma) and potassium ferrocyanide (. Gtoreq.98.5%, sigma-Aldrich). All solutions were prepared at 25 ℃ unless otherwise stated, using ultrapure water having a resistivity of 18.2 M.OMEGA.cm and stored at 4 ℃.

Making iEAT readers

Key chain detection (5.5X 2.5X 2.4 cm) ³ ) Built around a microcontroller unit (MCU, atamd 21G18, atmel Corporation). A digital-to-analog converter (DAC 8552, texas Instruments) is used to set the potential between the reference and working electrodes. For current measurement, a digital-to-analog converter (ADS 1115, texas Instruments) and potentiostat are connected to the peripheral interface of the MCU. The potentiostat comprises two operational amplifiers (AD 8608, analog Devices): one amplifier maintains a potential difference between the working electrode and the reference electrode, and the other amplifier acts as a transimpedance amplifier to convert the current into a voltage signal. Other peripheral devices include a communication module (bluetooth EZ-Link) for bluetooth connection with an external device, a display module, and a rechargeable battery.

Smart phone application

Using the MIT App Inventor 2, we created an Android application to facilitate system operation and data logging. The application allows the user to control the device, take a picture of the sample, and record measurement details (time point, current value, estimated analyte concentration, and GPS location). The data is stored in cloud storage (Google cloud hard disk).

Preparation of antibody-labeled Immunomagnetic beads (Ab-MBs)

Magnetic beads (. About.3.4X 10) ⁸ ) Resuspended in 1mL sodium phosphate buffer. The bead solution was vortexed briefly and the beads were collected by placing a magnet. The supernatant was discarded. This washing step was repeated twice. Next, the beads were mixed with about 100. Mu.g of capture or reference IgG and sodium phosphate buffer containing 1M ammonium sulfate. The total volume is 300. Mu.L so thatThe concentration of beads was 1.1X 10 ⁹ beads/mL. The bead solution was incubated at 4 ℃ for 16 hours with a slow inclined spin to avoid bead sedimentation. The surface epoxy groups of the magnetic beads allow direct covalent binding of the antibody via primary amino groups. The coated beads were then collected and washed three times with 1mL PBS. The prepared Ab-MB was then resuspended in 200. Mu.L PBS,1% BSA, and used as stock solution.

Freeze-drying of immunomagnetic beads and detection antibodies

Sucrose and PBS were used to prepare lyophilized Ab-MB and antibody. An amount of sucrose molarity (in PBS) 300 times higher than the antibody concentration or the same volume of PBS solvent (as sucrose was prepared) was added to the Ab-MB or antibody stock solution. The mixture was frozen in liquid nitrogen and then dried in a VirTis Freezemobile 25EL freeze-dryer (SP Scientific). The lyophilized reagents were stored at room temperature or 4 ℃ and reconstituted by the addition of 200 μ L of ultrapure distilled water prior to use.

Antigen standard

We used rice white flour as a model food substrate. 1.0g of rice flour was prepared in 10mL of PBS and boiled to prepare a well-mixed solution. We then added each of the five allergens (gliadin, ara h1, cor a1, casein, ovalbumin) to the rice solution.

Extraction buffer

Three extraction buffers were prepared: (1) 50mM Tris-HCl,150mM NaCl,

250mM 2-ME,2M GUA,1％SDS,pH 7.4；(2)50mM Tris-HCl,150mM NaCl,

20mM TCEP,2M GUA,pH 7.4；(3)50mM Tris-HCl,150mM NaCl,

5mM TCEP,2% N-lauroylsarcosine, pH7.4.

Food sample preparation

Food samples were from local supermarkets and restaurants. Food samples (about 1 g) were cut into small pieces and mixed with 19mL of extraction buffer. After allergen extraction, the supernatant was used as a sample extract for subsequent ifeat detection. The amount of allergen in the sample was restored by multiplying by 20-fold dilution.

iEAT assay

mu.L of the food extract was mixed with 50. Mu.L of Ab-MB solution and incubated for 3 minutes at room temperature. For washing, the beads were collected using a glass-encapsulated magnetic strip and released in PBS (100. Mu.L). The beads were then incubated with HRP-conjugated antibody (10. Mu.L) for 3 minutes and washed as described above. HRP-bead complex was mixed with TMB substrate and loaded onto the electrode. After 1 minute, chronoamperometric measurements were started. The current levels were averaged between 50 and 60 seconds.

Enzyme-linked immunosorbent assay (ELISA)

The stored capture and reference IgG antibodies were placed in carbonate-bicarbonate buffer (15 mM Na) ₂ CO ₃ ,35mM NaHCO ₃ pH 9.6) to-3. Mu.g/mL and added to 96-well polystyrene sterile flat-bottomed microplates (100. Mu.L/well) and incubated overnight at 4 ℃. The coated plate was washed three times with PBS (PBST) containing 0.05-after tween 20 to remove unbound antibody. PBS containing 1% BSA (100. Mu.L/well) was then added to cover the unoccupied binding sites. Plates were washed three times with PBST. Allergen standards and sample extracts (100 μ L/well) were dispensed in duplicate into wells and incubated for 1 hour at room temperature. Plates were washed three times with PBST. 100 μ L aliquots of biotinylated detection antibody were then added and incubated for 1 hour at room temperature. After three washes with PBST, fill with 100-fold diluted strept-HRP solution (100. Mu.L/well) for 30 minutes at room temperature. Plates were washed three times with PBST. The chemiluminescent signal was generated by adding 100. Mu.L of TMB substrate. After 10 minutes incubation at room temperature, 100. Mu.L of sulfuric acid (1N) was added to each well to stop the enzyme reaction. The optical absorbance of each well was measured at 450nm using a plate reader (TECAN).

Statistical analysis

All data obtained are expressed as mean ± Standard Deviation (SD). Statistical analysis was performed using GraphPad Prism 6. P-values below 0.05 were considered significant.

Discussion of the related Art

We have developed an instant food testing system (iEAT) that can sensitively detect multiple food antigens, test "safe" foods, and eliminate unnecessary avoidance, thereby providing support to consumers. We further envision the use of this system by the food industry, food reaction clinics and regulatory agencies. Signal detection based on electrochemical reactions is fast, scalable, well suited for compact electronics and for multiplexing. The key chain reader is prototype to be capable of operating independently, charging can be conducted in a wireless mode, and Bluetooth communication with cloud is achieved. The device is relatively inexpensive and the cost of measurement per antigen in current devices is approximately $ 3. With scale-up and the ability to produce lyophilized kits, we expect these costs to be greatly reduced. With these capabilities, the ifeat system is closely coupled to the world health organization's point-of-care device guide, known as accured, which is defined as being reasonably priced, sensitive, specific, user-friendly, fast and stable, equipment-free (i.e., without large power-dependent instruments), and deliverable.

We chose to quantify five representative model antigens, which are commonly present in consumer food products and are responsible for most food reactions. Although these antigens were chosen to validate the principle, there are many other potential antigens such as shellfish (shrimp, lobster), fin fish (tuna, salmon), nuts (walnuts, pecans, cashews), pollen and fruit, etc. It is relatively simple to add these and other allergens to the test list. The iEAT reader has single and multi-channel electrodes that can measure eight allergens simultaneously. Sequential measurement or expansion of the number of channels is a viable approach that allows for more extensive testing.

While we focus on specific protein antigens, current assay formats can also be modified by changing affinity ligands (e.g., aptamers, oligonucleotides) to detect small molecules, toxins, or nucleic acids; test panels for food safety (e.g., pesticides) and for food source identification (e.g., DNA-based testing) were created. The device may have many interesting applications, such as verifying food origin, confirming absence of contaminants or dietary restrictions to support religious purposes. These and other applications can be further enhanced by system integration, for example, by developing disposable fluidic devices to simplify sample processing. Regardless of the specific application, we envision that portable ifeat technology will allow more rigorous and proof-based analysis of foods, enhance consumer protection, reduce accidental allergy exposure, and identify problems in our food supply chain.

Other embodiments

While this specification contains many specifics, these should not be construed as limitations on the scope of what may be claimed, but rather as descriptions of features specific to particular examples. Some features that are described in this specification in the context of separate embodiments may also be combined. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims (22)

1. A target analyte detection device comprising:

a housing containing a potentiostat and a microcontroller coupled to the potentiostat;

a substrate comprising a plurality of electrodes on a first surface of the substrate, wherein a first set of electrodes of the plurality of electrodes define a first sample detection region, wherein the substrate is removably attached to the housing such that the first set of electrodes is coupled to the potentiostat when the substrate is attached to the housing; and

a magnet assembly coupleable to the second surface of the substrate, wherein the magnet assembly comprises a magnet positioned in the magnet assembly such that, upon coupling the magnet assembly to the substrate, a magnetic field from the magnet extends through the substrate and the first set of electrodes into a region above the first specimen detection region;

wherein the housing further comprises:

a digital-to-analog converter circuit, wherein an output of the digital-to-analog converter circuit is electrically coupled to an input of the potentiostat;

an analog-to-digital converter circuit, wherein an input of the analog-to-digital converter circuit is coupled to an output of the potentiostat; and

a microcontroller electrically coupled to the digital-to-analog converter circuit and the analog-to-digital converter circuit, wherein the microcontroller is configured to provide a voltage signal to an input of the digital-to-analog converter circuit, and wherein the microcontroller is configured to receive a measurement signal from an output of the analog-to-digital converter circuit;

the first sample detection region has a sample applied thereto, comprising: a target analyte having different kinds of first and second markers, a plurality of magnetic beads including first binding moieties that specifically bind to the first markers of the target analyte, second binding moieties that bind to the second markers of the target analyte, an active enzyme that binds to the second binding moieties, and an electron mediator solution;

the first set of electrodes comprises a reference electrode, a counter electrode, and a working electrode;

the potentiostat is electrically coupled to each electrode of the first set of electrodes and is configured to maintain a predetermined potential difference between the working electrode and the reference electrode during operation to induce a redox reaction between the active enzyme and the electron mediator such that the redox reaction occurs and a current is generated from the working electrode to the counter electrode, and the potentiostat is further configured to simultaneously measure the current from the working electrode to the counter electrode during operation;

the potentiostat comprises a first operational amplifier electrically configured to maintain a predetermined potential difference between the working electrode and the reference electrode, and a second operational amplifier electrically configured to convert a current from the working electrode to the counter electrode into a voltage signal;

the analog-to-digital converter circuit digitizes the voltage signal and transmits the digitized voltage signal to the microcontroller for processing to determine a specific characteristic of the target analyte;

the microcontroller is further configured to transmit a digital control signal to the digital-to-analog converter circuit and to convert the digital control signal by the digital-to-analog converter circuit into a corresponding analog signal for increasing and/or decreasing the potential of the working electrode relative to the reference electrode and to control the sampling of current from the working electrode to the counter electrode by applying the analog signals to the inputs of the first and second operational amplifiers.

2. The apparatus of claim 1, further comprising a plurality of potentiostats:

wherein the plurality of electrodes includes at least one set of additional electrodes, each set of electrodes being coupled to a different respective potentiostat of the plurality of potentiostats, and each set of electrodes defining a different respective sample detection area.

3. The apparatus of claim 2, comprising a multiplexer electrically coupled to an output of each potentiostat of the plurality of potentiostats, wherein the multiplexer is configured to electrically couple a selected output to an input of the analog-to-digital converter circuit.

4. The device of claim 2, comprising a well plate disposed on a substrate surface, the well plate having a plurality of wells, wherein each well of the plurality of wells can be disposed directly on a different respective sample detection region.

5. The apparatus of claim 2, wherein the magnet assembly comprises a plurality of magnets, and wherein, when the magnet assembly is coupled to the substrate, each magnet of the plurality of magnets is positionable adjacent the substrate and in alignment with a respective set of electrodes such that the magnetic field extends from the magnet through the substrate and the respective set of electrodes into a region above a sample detection region defined by the respective set of electrodes.

6. The device of claim 1, wherein the substrate comprises a card edge connector and the device further comprises a card edge connector receptacle.

7. The apparatus of claim 1, wherein the apparatus further comprises an electronic communication interface.

8. The apparatus of claim 7, wherein the electronic communication interface comprises at least one of a universal serial bus connector or a wireless transceiver.

9. The device of claim 1, wherein the first set of electrodes comprises three separate electrodes.

10. The device of claim 9, wherein a first electrode and a second electrode of the three separate electrodes are a first metal and a third electrode of the three separate electrodes is a second metal.

11. The device of claim 1, wherein the first set of electrodes consists of two separate electrodes.

12. The device of claim 1, wherein the first set of electrodes comprises interdigitated electrodes.

13. A method of detecting the presence of a target analyte in a first fluid sample, comprising:

providing a plurality of magnetic beads to a first fluid sample, wherein the first fluid sample comprises a target analyte carrying a first marker and a second marker of different species, the plurality of magnetic beads comprises a first binding moiety that specifically binds to the target analyte, and the first binding moiety binds to the first marker carried on the target analyte;

binding the plurality of magnetic beads to a target analyte in a first fluid sample;

transferring the magnetic beads from the first fluid sample to a second fluid sample, wherein the second fluid sample comprises a second binding moiety that specifically binds to the target analyte, and wherein the second binding moiety is bound to an active enzyme,

allowing the second binding moiety in the second fluid sample to bind to the target analyte bound to the first binding moiety of the magnetic bead and the second binding moiety to bind to a second marker carried on the target analyte;

combining a second fluid sample comprising a plurality of magnetic beads and a second binding moiety with an electron mediator solution to obtain a third fluid sample;

providing the third fluid sample to a sample detection region of the substrate, wherein the sample detection region is disposed on the first set of electrodes and the first set of electrodes is electrically coupled to a potentiostat;

inducing a redox reaction between the electron mediator and the active enzyme in the third fluid sample, wherein the signal generated by a first potentiostat of the plurality of potentiostats is derived from the redox reaction;

monitoring a plurality of outputs of a plurality of potentiostats to determine the presence of a target analyte,

wherein monitoring the plurality of outputs of the plurality of potentiostats comprises:

the outputs of the potentiostats are polled simultaneously,

selecting measurement data from one or more of a plurality of outputs of the plurality of potentiostats;

analyzing, by a microcontroller, the selected measurement data to obtain data regarding the analysis of the third fluid sample; and

outputting data from the microcontroller regarding the analysis of the third fluid sample to a display;

the first set of electrodes comprises a reference electrode, a counter electrode, and a working electrode;

electrically coupling the potentiostat to each electrode of the first set of electrodes and configured to maintain a predetermined potential difference between the working electrode and the reference electrode during operation to induce a redox reaction such that the redox reaction occurs and a current is generated from the working electrode to the counter electrode, and to simultaneously measure the current from the working electrode to the counter electrode during operation;

the potentiostat comprises a first operational amplifier electrically configured to maintain a predetermined potential difference between the working electrode and the reference electrode, and a second operational amplifier electrically configured to convert a current from the working electrode to the counter electrode into a voltage signal;

an analog-to-digital converter circuit digitizes the voltage signal and transmits the digitized voltage signal to the microcontroller for processing to determine a specific characteristic of the target analyte;

the microcontroller is configured to transmit a digital control signal to the digital-to-analog converter circuit and to convert the digital control signal by the digital-to-analog converter circuit into a corresponding analog signal for increasing and/or decreasing the potential of the working electrode relative to the reference electrode and to control the sampling of current from the working electrode to the counter electrode by applying the analog signals to the inputs of the first and second operational amplifiers.

14. The method of claim 13, wherein simultaneously polling the plurality of outputs of the plurality of potentiostats comprises, for each potentiostat of the plurality of potentiostats, measuring a voltage or current from a respective electrode coupled to the potentiostat, wherein the voltage or current from the respective electrode is indicative of the presence and/or prevalence of the analyte of interest in a respective sample detection region associated with the potentiostat.

15. The method of claim 13, wherein the active enzyme comprises horseradish peroxidase (HRP) and the electron mediator solution comprises 3,3', 5' -Tetramethylbenzidine (TMB).

16. The method of claim 13, wherein the target analyte comprises an extracellular vesicle.

17. The method of claim 16, wherein extracellular vesicles comprise exosomes.

18. The method of any of claims 16-17, further comprising:

comparing the output of the potentiostat to a reference level to determine whether the output is above or below the reference level; and is

An amount of increase or decrease in the target analyte is determined based on the comparison.

19. The method of claim 13, wherein the target analyte is loaded with one or more immune cell markers.

20. The method of claim 13, wherein the first fluid sample comprises blood or urine.

21. The method of claim 13, wherein target analytes comprise proteins, cells, peptides, lipids, toxins, nucleic acids, microorganisms, food antigens, or metabolites.

22. The method of claim 13, further comprising exposing the third fluid sample to a magnetic field to maintain a plurality of magnetic beads in the third fluid sample in a position proximate to a first set of electrodes, wherein the first set of electrodes is electrically coupled to a first potentiostat.

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